High molecular weight polysaccharide that binds and inhibits virus

ABSTRACT

This invention provides a high molecular weight polysaccharide capable of binding to and inhibiting virus and related pharmaceutical formulations and methods of inhibiting viral infectivity and/or pathogenicity, as well as immunogenic compositions. The invention further includes methods of inhibiting the growth of cancer cells and of ameliorating a symptom of aging. Additionally, the invention provides methods of detecting and/or quantifying and/or isolating viruses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. non-provisional applicationSer. No. 14/107,553, filed on Dec. 16, 2013, which is a continuation ofU.S. non-provisional application Ser. No. 12/694,226, filed on Jan. 26,2010, U.S. Pat. No. 8,629,121, issued Jan. 14, 2014, which claims thebenefit of U.S. provisional application No. 61/206,197, filed Jan. 27,2009, all of which are hereby incorporated by reference in theirentirety (including the provisional application's appendix).

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant no.DE-FG09-93 ER-20097 awarded by the Department of Energy-funded Centerfor Plant and Microbial Complex Carbohydrates. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention relates to a high molecular weight polysaccharide capableof binding to and inhibiting virus and related immunogenic compositionsand methods.

BACKGROUND OF THE INVENTION

Extracts prepared from grape seed have been touted as having numeroushealth benefits, including antioxidant, anti-inflammatory,anti-bacterial, anti-viral, and anti-tumor activities. At least some ofthese effects have been attributed to flavonoid constituents of a hotwater extract of grape seed. Nair et al., Clin Diagn Lab Immunol. (March2002) 9(2):470-476.

SUMMARY OF THE INVENTION

In certain embodiments, the invention provides a composition that bindsto a virus and inhibits the virus, the composition including:

-   -   an isolated polysaccharide comprising an arabinofuranosyl        residue, a galactopyranosyl residue, and a galactouronic acid;        and    -   a catechin polymer;    -   wherein said composition is soluble in water; and        wherein said composition binds to a virus and reduces the        infectivity or pathogenecity of said virus.

In certain embodiments, the invention provides a composition that bindsto a virus and inhibits the virus, the composition including:

-   -   an isolated polysaccharide including a arabinofuranosyl residue,        a rhamnopyranosyl residue, a galactopyranosyl residue, a        glucopyranosyl residue, a mannopyranosyl residue, and a        galactouronic acid; and    -   a non-carbohydrate aromatic polymer;    -   wherein the composition includes about 40 to about 44 percent        oxygen, about 44 to about 48 percent carbon, about 3 to about 6        percent hydrogen; and about 0.1 to about 1 percent nitrogen;    -   wherein the composition is soluble in water; and    -   wherein the composition binds to a virus and reduces the        infectivity or pathogenecity of the virus.

The composition can include about 10 to about 30 weight percentpolysaccharide and about 70 to about 90 weight percent catechin polymer.Although such a composition is less than 50 weight percentpolysaccharide, this composition is still referred to herein as a“polysaccharide composition” or “polysaccharide” for ease ofdescription.

In certain embodiments, the composition includes a component having amolecular weight greater than about 1 million Daltons. In certainembodiments, the composition includes a component having a molecularweight in the range of about 60,000 to about 100,000 Daltons. In someembodiments, the composition includes both components. In variations ofthese embodiments, the ratio of the amount of the component having amolecular weight greater than about 1 million Daltons to the amount ofthe component having a molecular weight in the range of about 60,000 toabout 100,000 Daltons is about 95:5.

In particular embodiments, the composition does not include protein.

In exemplary compositions, the polysaccharide can include:

-   -   about 30 to about 75 mole percent arabinose;    -   about 0 to about 10 mole percent rhamnose;    -   about 0 to about 5 mole percent xylose;    -   about 0 to about 8 mole percent glucuronic acid;    -   about 3 to about 36 mole percent galactouronic acid;    -   about 0 to about 6 mole percent mannose;    -   about 1 to about 20 mole percent galactose; and    -   about 0 to about 13 mole percent glucose.

In other exemplary compositions, the polysaccharide can include:

-   -   about 60 to about 66 mole percent arabinose;    -   about 4.1 to about 4.5 mole percent rhamnose;    -   about 2.2 to about 2.4 mole percent xylose;    -   about 8.7 to about 9.7 mole percent galactouronic acid;    -   about 2.3 to about 2.5 mole percent mannose;    -   about 13.7 to about 15.1 mole percent galactose; and    -   about 4.2 to about 4.6 mole percent glucose.

In certain embodiments, the compositions include:

-   -   a terminally linked arabinofuranosyl residue (t-Araf);    -   a 2-linked arabinofuranosyl residue (2-Araf);    -   a 2-linked rhamnopyranosyl residue (2-Rhap);    -   a 3-linked arabinofuranosyl residue (3-Araf);    -   a terminally linked galactopyranosyl residue (t-Gal);    -   a 5-linked arabinofuranosyl residue (5-Araf);    -   a 3-linked glucopyranosyl residue (3-Glc) and/or a 2,4-linked        rhamnopyranosyl residue (2,4-Rhap);    -   a 2-linked glucopyranosyl residue (2-Glc);    -   a 4-linked mannopyranosyl residue (4-Man);    -   a 3,5-linked arabinofuranosyl residue (3,5-Araf);    -   a 2,5-linked arabinofuranosyl residue (2,5-Araf);    -   a 4-linked glucopyranosyl residue (4-Glc);    -   a 2,3,5-linked arabinofuranosyl residue (3,5-Araf) and/or a        2,3,4-linked arabinopyranosyl residue (2,3,4-Arap);    -   a 4-linked galactouronic acid (4-gal A);    -   a 3,6-linked galactopyranosyl residue (3,6-Gal);    -   a 2,3,4,6-linked mannopyranosyl residue (2,3,4,6-Man);    -   a 2,3,4,6-linked galactopyranosyl residue        (2,3,4,6-Gal)&2,3,4-linked galactouronic acid; and    -   a 2,3,4,6-linked glucopyranosyl residue (2,3,4,6-Glc).        In specific examples of such embodiments, the polysaccharide        includes:    -   the terminally linked arabinofuranosyl residue (t-Araf) includes        about 14.2 to about 15.7 wt percent of the polysaccharide;    -   the 2-linked arabinofuranosyl residue (2-Araf) includes about        9.1 to about 10.08 wt percent of the polysaccharide;    -   the 2-linked rhamnopyranosyl residue (2-Rhap) includes about 0.3        wt percent of the polysaccharide;    -   the 3-linked arabinofuranosyl residue (3-Araf) includes about        3.0 to about 3.4 wt percent of the polysaccharide;    -   the terminally linked galactopyranosyl residue (t-Gal) includes        about 2.0 to about 2.2 wt percent of the polysaccharide;    -   the 5-linked arabinofuranosyl residue (5-Araf) includes about        15.0 to about 16.6 wt percent of the polysaccharide;    -   the 3-linked glucopyranosyl residue (3-Glc) and/or 2,4-linked        rhamnopyranosyl residue (2,4-Rhap) includes about 0.7 wt percent        of the polysaccharide;    -   the 2-linked glucopyranosyl residue (2-Glc) includes about 1.1        to about 1.3 wt percent of the polysaccharide;    -   the 4-linked mannopyranosyl residue (4-Man) includes about 1.3        to about 1.5 wt percent of the polysaccharide;    -   the 3,5-linked arabinofuranosyl residue (3,5-Araf) includes        about 6.6 to about 7.3 wt percent of the polysaccharide;    -   the 2,5-linked arabinofuranosyl residue (2,5-Araf) includes        about 5.0 to about 5.6 wt percent of the polysaccharide;    -   the 4-linked glucopyranosyl residue (4-Glc) includes about 4.6        to about 5.0 wt percent of the polysaccharide;    -   the 2,3,5-linked arabinofuranosyl residue (3,5-Araf) and/or        2,3,4-linked arabinopyranosyl residue (2,3,4-Arap) includes        about 25.7 to about 28.4 wt percent of the polysaccharide;    -   the 4-linked galactouronic acid (4-gal A) includes about 1.4 to        about 1.6 wt percent of the polysaccharide;    -   the 3,6-linked galactopyranosyl residue (3,6-Gal) includes about        0.4 wt percent of the polysaccharide;    -   the 2,3,4,6-linked mannopyranosyl residue (2,3,4,6-Man) includes        about 0.7 wt percent of the polysaccharide;    -   the 2,3,4,6-linked galactopyranosyl residue        (2,3,4,6-Gal)&2,3,4-linked galactouronic acid includes about 2.0        to about 2.2 wt percent of the polysaccharide; and    -   the 2,3,4,6-linked glucopyranosyl residue (2,3,4,6-Glc) includes        about 2.1 to about 2.3 wt percent of the polysaccharide.

Polysaccharide compositions according to the invention can bind anyvirus, including, but not limited to a virus from a family selected fromthe group consisting of Adenoviridae, Picornaviridae, Reoviridae,Arenaviridae, Bunyaviridae, Coroanviridae, Herpesviridae,Orthomyxoviridae, Paramyxoviridae, Poxviridae Rhabdoviridae,Flaviviridae, and/or Retroviridae. In certain embodiments, thecomposition provides a NMR spectrum as shown in FIG. 3A.

Polysaccharide compositions can be formulated in a pharmaceuticallyacceptable excipient for administration to a subject. In particularembodiments, the composition is formulated as a unit dosage formulation.For example, the composition can be formulated in a delivery formselected from the group consisting of a tablet, a capsule, a lozenge, anointment, a cream, a transdermal formulation (e.g., a patch), a gel, anasal spray, a suppository, an injectable, and an implantablesustained-release formulation.

The invention also provides a method of preparing a composition thatbinds a virus. The method entails preparing a substantially homogeneousaqueous mixture or solution of plant material from one or more plants ofthe Vitaceae family; contacting the mixture with an ion exchange resinand recovering the colored product; and further purifying the coloredproduct by removing low molecular weight components that can passthrough a dialysis filter that generally passes molecules having amolecular weight of a 5×10⁵ Daltons or less to produce a compositionthat binds a virus. The plant material can be from a grape plant, e.g.,the plant material can include seed from a grape plant. In certainembodiments, the ion exchange resin includes an anion exchange resin. Invariations of such embodiments, the method entails running the mixturethrough two sequential anion exchange columns. Further purification ofthe colored product can entail dialyzing or ultrafiltering the coloredproduct. In certain embodiments, the further purification increases theconcentration of the colored product by at least 10 fold. Thus, in anexemplary method, the ion exchange resin includes an anion exchangeresin; and the further purification includes ultrafiltering or dialyzingthe colored product to increase the concentration of the colored productby at least 10 fold. The invention also provides a composition thatbinds virus, wherein the composition is obtainable, or obtained, by anyof the above-described preparation methods of the invention.

Another aspect of the invention is a method of inhibiting theinfectivity and/or pathogenicity of a virus, the method includingcontacting the virus with a polysaccharide composition according to theinvention. The virus can be any of those described above with respect tothe binding characteristics of the polysaccharide compositions accordingto the invention. In particular embodiments, the composition isconveniently provided in a unit dosage form. In exemplary embodiments,the composition is administered to a mammal, such as, e.g., a human inneed thereof. Administration can be by any route, such as oraladministration, sub-lingual administration, topical administration,transdermal administration, nasal administration, rectal administration,injectable administration, and administration via an implant.

The invention additionally provides a method inhibiting the growthand/or proliferation of a cancer cell, by contacting the cancer cellwith a polysaccharide composition of the invention. The cancer cell canbe, for example, a solid tumor cell or a non-solid tumor cell. Incertain embodiments, the cancer cell is a metastatic cancer cell. Inparticular embodiments, the cancer is bladder cancer, breast cancer,colon and/or rectal cancer, endometrial cancer, kidney cancer, leukemia,liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, ovariancancer, pancreatic cancer, prostate cancer, skin cancer (non-melanoma),stomach cancer, or thyroid cancer. In certain embodiments, thecomposition is conveniently provided in a unit dosage form. In exemplaryembodiments, the composition is administered to a mammal, such as, e.g.,a human in need thereof. Administration can be by any route, such asoral administration, sub-lingual administration, topical administration,transdermal administration, nasal administration, rectal administration,injectable administration, and administration via an implant.

Another aspect of the invention is an immunogenic composition, whereinthe composition includes a polysaccharide composition of the inventionbound to a virus, such as one from a family selected from: Adenoviridae,Picornaviridae, Reoviridae, Arenaviridae, Bunyaviridae, Coroanviridae,Herpesviridae, Orthomyxoviridae, Paramyxoviridae, PoxviridaeRhabdoviridae, Flaviviridae, and Retroviridae. In particularembodiments, the immunogenic composition includes an adjuvant, such as,e.g., alum.

The invention also provides a method of inducing an immune response in amammal. The method entails administering to the mammal an immunogeniccomposition according to the invention in an amount sufficient to inducean immune response. The immune response can, for example, be directedagainst one or more viruses from any of the families noted above. Inspecific embodiments, the method can include preparing monoclonalantibodies that bind to any of these viruses.

Another aspect of the invention is a method of ameliorating a symptom ofaging in a subject in need thereof. The method entails administering tothe subject an effective amount of a polysaccharide compositionaccording to the invention, wherein said effective amount is sufficientto ameliorate a least one symptom of aging. Illustrative symptoms ofaging that can be ameliorated by this method include hair loss, nearvision loss due to aging, age spots, skin thinning and/or loss ofelasticity, reduced hormone levels due to aging, loss of stamina due toaging, increased fat deposits, chronic joint and back pains, and/orage-related impairments in cognition. In particular embodiments, thecomposition is conveniently provided in a unit dosage form. In exemplaryembodiments, the composition is administered to a mammal, such as, e.g.,a human in need thereof. Administration can be by any route, such asoral administration, sub-lingual administration, topical administration,transdermal administration, nasal administration, rectal administration,injectable administration, and administration via an implant.

Another aspect of the invention is a method of detecting and/orquantifying a virus. This method entails contacting a sample with apolysaccharide composition of the invention under conditions suitablefor the polysaccharide composition to bind any virus present in thesample; and detecting and/or quantifying binding to the polysaccharidecomposition.

The invention also provides a device that can be used in the detectionmethod of the invention. The device includes a solid support havingattached thereto a polysaccharide composition of the invention.Exemplary solid supports include a planar substrate, a bead, acapillary, a microchannel, and a syringe. In particular embodiments, thesolid support can include a material selected from the group consistingof quartz, glass, plastic, metal, gel, and an aerogel.

Another aspect of the invention is a method of isolating one or moreviruses from a sample. The method entails contacting the sample with thepolysaccharide composition of the invention that is affixed to asubstrate under conditions suitable for the polysaccharide compositionto bind any virus present in the sample. The substrate is then washed toremove any unbound sample material.

In other embodiments, a polysaccharide composition described herein canbe administered to a subject to ameliorate and/or reverse a symptom ofaging. Symptoms of aging that can be ameliorated and/or reversed by thismethod include, but are not limited to, hair loss, near vision loss dueto aging, age (“liver”) spots, reduced hormone (e.g. estrogen ortestosterone) levels due to aging, loss of stamina due to aging,increased fat deposits, chronic joint and back pains, age-relatedimpairments in cognition and focus of thinking.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a summary of the effects of three different concentrationsof polysaccharide composition on virus infectivity. See Example 6. “LFT”refers to the polysaccharide composition of the invention prepared asdescribed in Example 1. In each triplet of bars for each virus, the topbar is undiluted LFT, the middle bar is LFT 1:10, and the bottom bar isLFT 1:100.

FIG. 2A-C shows three HPLC chromatograms that indicate that, when thepolysaccharide composition binds virus, a component of the compositionis released that is approximately 180,000 Daltons, and a largermolecular weight component remains bound to the virus.

FIG. 3A-E shows NMR of a polysaccharide composition of the invention. 3Ashows the 1-D proton spectrum; 3B-3D shows 2-D gradient enhanced COSY,TOCSY and NOESY NMR spectra, respectively; 3E shows the NMR spectrum ofthe non-carbohydrate portion of the polysaccharide composition.

FIG. 4A-C are photographs showing the effect of treating a humansquamous cell carcinoma with a polysaccharide composition of theinvention as described in Example 10. A shows the lesion as it appearedbefore treatment; B shows the lesion on treatment day 5; and C shows thelesion on treatment day 9. The blue dot in each photograph is ¼ inch indiameter.

FIG. 5A-B are photographs showing the effect of treating a human skincancer in a male subject by oral administration of a polysaccharidecomposition of the invention as described in Example 11. A shows thelesion as it appeared before treatment; B shows the lesion after 20 oraltreatments. The blue dot in each photograph is ¼ inch in diameter.

FIG. 6A-C are photographs showing the effect of treating a humansquamous cell carcinoma in a female subject by oral administration of apolysaccharide composition of the invention as described in Example 11.A shows the lesion as it appeared on the morning of 7-27-08 (12 hourspost-treatment); B shows the lesion in the evening of 7-28-08 (36 hourspost-treatment); C shows the lesion on 8-1-08 (day 7 post-treatment).

FIGS. 7A-B are photographs showing hair restoration by oraladministration of a polysaccharide composition of the invention to an89-year-old male subject as described in Example 13. The picture in Awas taken 2 weeks before the picture in B.

FIG. 8 shows the results of Example 14: Size Exclusion Chromatography.The filtered polysaccharide was purified on a HW 65 column, and 2-minfractions were collected. Elution was monitored by refractive indexdetection which is not specific for carbohydrate. However, it isestimated to be below 5 kD and made of smaller oligosaccharides.

FIG. 9 shows the results of Example 14: 1-D Proton NMR Spectrum ofGalahad Lot 1 (CCRC Code DK121806). A polysaccharide compositionaccording to the invention is referred to as “Galahad” in Example 14. Acomponent of the polysaccharide composition is a red material that canbe precipitated by addition of ammonium sulfate and can thus beseparated from the carbohydrate in the composition. This red material isreferred to as “Galahad Red” in this example.

FIG. 10 shows the results of Example 14: Partial 2-D COSY NMR Spectrumof Galahad Lot 1 (CCRC Code DK121806).

FIG. 11 shows the results of Example 14: 2-D HSQC NMR Spectrum ofGalahad Lot 1 (CCRC Code DK121806).

FIG. 12 shows the results of Example 14: Dextran standard curve.

FIG. 13 shows the results of Example 14: TLC Analysis of Galahad andGreen Tea Extracts as well as Galahad butanolic HCl iron digest. Lane1—Catechin 15 ug; Lane 2—Green Tea Extract 20 ug; Lane 3—Galahad Ethylacetate Extract, 40 ug; Lane 4—Galahad butanolic HCl iron digest; Lane5—Proanthocyanidin B1 (dimeric proanthocyanidin), 20 ug.

FIG. 14 shows the results o Example 14: Analysis of Intact Galahad andPhloroglucinol Products. TLC Plate at left: the ammonium sulfateprecipitate and DEAE purified material (lanes 1 and 2 respectively) incomparison to 2 μl of Carlo Rossi burgundy which has a variety ofanthocyanins and proanthocyanins. Considerable staining is observed atthe origin of both Galahad lanes, consistent with a high molecularweight polymer. Middle and right TLC plates: oroglucinol reactionproducts. Lane 1—Catechin, 10 μg; Lane 2—Carlo Rossi Burgundy, Lane3—Ammonium Sulfate precipitate; Lane 4 DEAE Purified Galahad; Lane 5Phloroglucinol, 4 ug. Middle plate is developed in chloroform:methanol9:1 to look for anthocyanidin adducts. Right plate is developed inchloroform:methanol: aqueous KCl 5:4:1 to look for partial degradationproducts. Note the series of slow eluting bands in the right plate seenin A. S. ppt and Carlo Rossi Burgundy. These may be partial breakdownproducts.

FIG. 15 shows the results of Example 14: MALDI TOF Analysis of IatrobeadPool 2 from Phloroglucinolysis Reaction 2. Mass at m/z 342 is consistentwith the sodium adduct of peonidin or petunidin.

FIG. 16 shows the results of Example 14: TLC of PhloroglucinolysisReaction Mix and Fractions from C18 Column. Phloroglucinolysis of abatch of Galahad Red produced an array of compounds as can be seen inlane 2 of each TLC. Lane 1 of each TLC corresponds to phloroglucinol,which is eluted in the load and 5% acetonitrile wash of the column, asshown in lanes 3, 4, and 5 of the TLC on the left. Methylatedphloroglucinol elutes in the 15% and early 100% acetonitrile washes(lane 8 right plate and lane 7 of left plate) while most of thecompounds produced on phloroglucinolysis elute with 100% acetonitrile(lanes 7 and 8 left plate). Benzene isopropanol elutes a strongly yellowband (lane 5 right plate) while addition of TFA to this solvent elutes ared band that remains at the origin (lane 7, right plate). Circled bandsare visible without stain. Letters indicate color; P for pink, Y foryellow and B for brown.

FIG. 17 shows the results of Example 14: TLC of Silica Gel Purificationof Acetonitrile Wash from C18. Acetonitrile eluate 2 from C18chromatography was purified by preparative TLC. Shown are the reactionmix (lane 1) followed by the 7 pools (numbered). Circled bands arevisible without stain and labeled as in FIG. 16.

FIG. 18 shows the MALDI/MS results of Example 14: Silica Pool 1—PositiveMode.

FIG. 19 shows the MALDI/MS results of Example 14: Silica Pool 2—PositiveMode.

FIG. 20 shows the MALDI/MS results of Example 14: Silica Pool 3—PositiveMode.

FIG. 21 shows the MALDI/MS results of Example 14: Silica Pool 4—PositiveMode.

FIG. 22 shows the MALDI/MS results of Example 14: Silica Pool 6—PositiveMode.

FIG. 23 shows the MALDI/MS results of Example 14: Silica Pool 7—PositiveMode.

FIG. 24 shows the MALDI/MS results of Example 14: Benzene IsopropanolEluate—Positive Mode.

FIG. 25 shows the MALDI/MS results of Example 14: Benzene IsopropanolTFA—Positive Mode.

FIG. 26 shows the HPLC results of Example 14: Phloroglucinolysis BlankReaction.

FIG. 27 shows the HPLC results of Example 14: Catechin Standard.

FIG. 28 shows the HPLC results of Example 14: Proanthocyanidin B1—Notethat injection volume was 200 ul of 20% methanol, accounting for thepeak shape of the catechin-phloroglucinol peak at 14.4 min.

FIG. 29 shows the HPLC results of Example 14: Galahad 20 min reaction.

FIG. 30 shows the HPLC results of Example 14: Galahad 60 Min Reaction.

FIG. 31 shows the HPLC results of Example 14: 60 minute Reaction 200 ulinjection for MS.

FIG. 32 shows the HPLC results of Example 14: Green Tea Pool 2.

FIG. 33 shows the HPLC results of Example 14: Green Tea Pool 3.

FIG. 34 shows the NMR spectroscopy results of Example 14: 1D proton NMRspectrum of GH-C18-1, a fraction from Sep-Pak C18 eluted with 10 mMammonium acetate and water. Aromatic compound content is low in thisfraction. The carbohydrates are O-methylated as indicated by the signalsat 3.82 ppm.

FIG. 35 shows the NMR spectroscopy results of Example 14: 1D proton NMRspectrum of GH-C18-2, a fraction from Sep-Pak C18 eluted withacetonitrile and dissolved in methanol-d4. Aromatic compound content ismuch higher in this fraction than in GH-C18-1, and carbohydrate contentis significantly lower.

FIG. 36 shows the NMR spectroscopy results of Example 14: 1D proton NMRspectrum of GH-C18-3, a fraction from Sep-Pak C18 eluted withacetonitrile and dissolved in D2O. The molecular weight of thecarbohydrate in this fraction is lower as compared with the other two,GH-C18-1 and GH-C18-2. The carbohydrate is most likely arabinan withdifferent linkages.

FIG. 37 shows the NMR spectroscopy results of Example 14: 1D proton NMRspectrum of GH-alcohol-1, a fraction precipitated in 90% MeOH anddissolved in D2O. Aromatic compound content is low in this fraction. Thecarbohydrates are O-methylated as indicated by the signals at 3.81 ppm.This fraction is very similar to GH-C18-1 in composition.

FIG. 38 shows the NMR spectroscopy results of Example 14: 1D proton NMRspectrum of GH-alcohol-2, a fraction precipitated in 80% iso-propanoland dissolved in D2O. The molecular weight of the carbohydrate in thisfraction is lower as indicated by the sharp peaks. Again, thecarbohydrate is most likely arabinan with different linkages. Thisfraction is very similar to GH-C18-3 in composition.

FIG. 39 shows the NMR spectroscopy results of Example 14: 1D proton NMRspectrum of GH-alcohol-3, a fraction soluble in 80% iso-propanol anddissolved in D2O. Very similar to GH-C18-2, aromatic compound content ismuch higher in this fraction than in GH-alcohol-1 or GH-alcohol-2, andcarbohydrate content is significantly lower, which is consistent withthe results from GC analysis.

FIG. 40 shows the structure of an illustrative procyadin polymer.

DETAILED DESCRIPTION

The invention relates to the discovery of a complex composition thatconsists of sugars and an aromatic polymer that can be obtained fromplants, such as those in the genus Vitis, particularly the familyVitaceae, which are a family of dicotyledonous flowering plantsincluding the grape and Virginia creeper. This composition hasanti-viral and anti-cancer activities. The composition binds to virusesand virus-like structure, e.g., structure containing nucleic acidsurrounded by a protein coat that protects the nucleic acid; and mayhave an envelope (e.g., a lipid envelope) that surrounds the proteincoat. Virus-like structures can include non-replicating and/orartificial viruses. The properties and use of the composition arediscussed herein with respect to viruses for convenience, but those ofskill in the art understand that these properties and many uses alsoapply to virus-like particles. The composition can be contacted with avirus to produce an immunogenic composition that induces an immuneresponse against the virus. Viral binding can also be exploited todetect, isolate, filter, label, bind, inactivate, identify or quantifyviruses.

I. High Molecular Weight Polysaccharide Composition

A. Characteristics

The composition of the invention (“polysaccharide composition”) ischaracterized by a high molecular weight polysaccharide that is isolatedby separating the composition from at least one other component(s) thatis/are naturally present with the polysaccharide. One way of carryingout this separation is by passing aqueous mixture or solution of plantmaterial from one or more plants of the Vitaceae family over an ionexchange resin and recovering the colored product. See Example 1.

In particular embodiments, isolated polysaccharide composition includesan arabinofuranosyl residue, a galactopyranosyl residue, and agalactouronic acid. The polysaccharide composition also includes acatechin polymer. In variations of these embodiments, the catechinpolymer accounts for about 70 to about 90 weight percent of thecomposition, with about 10 to about 30 weight percent beingpolysaccharide.

In certain embodiments, the isolated polysaccharide composition includesan arabinofuranosyl residue, a rhamnopyranosyl residue, agalactopyranosyl residue, a glucopyranosyl residue, a mannopyranosylresidue, and a galactouronic acid. See Example 3. The polysaccharidecomposition also includes a non-carbohydrate aromatic polymer. SeeExample 2. Elemental analysis indicates that the composition comprisesabout 40 to about 44 percent oxygen, about 44 to about 48 percentcarbon, about 3 to about 6 percent hydrogen, and about 0.1 to about 1percent nitrogen. In one embodiment, the approximate elementalcomposition is 41.73 percent oxygen, 45.55 percent carbon, 4.48 percenthydrogen, and 0.37 percent nitrogen.

In particular embodiments, the polysaccharide composition comprises acomponent having a molecular weight greater than about 0.05 millionDaltons and typically less than about 5 million Daltons. In certainembodiments, the composition includes a component having a molecularweight greater than about 1 million Daltons. In certain embodiments, thecomposition includes a component having a molecular weight in the rangeof about 60,000 to about 100,000 Daltons. In some embodiments, thecomposition includes both components. In variations of theseembodiments, the ratio of the amount of the component having a molecularweight greater than about 1 million Daltons to the amount of thecomponent having a molecular weight in the range of about 60,000 toabout 100,000 Daltons is about 95:5. The polysaccharide compositiongenerally does not contain protein, as determined by Bradford assay.

In exemplary compositions, the polysaccharide can include:

about 30 to about 75 mole percent arabinose;

about 0 to about 10 mole percent rhamnose;

about 0 to about 5 mole percent xylose;

about 0 to about 8 mole percent glucuronic acid;

about 3 to about 36 mole percent galactouronic acid;

about 0 to about 6 mole percent mannose;

about 1 to about 20 mole percent galactose; and

about 0 to about 13 mole percent glucose.

In particular embodiments, the polysaccharide composition is typicallybetween about 7.5 and about 9.0 weight percent total carbohydrate, morespecifically about 8 percent total carbohydrate. In certain embodiments,the glycosyl composition of the polysaccharide composition of theinvention is:

about 60 to about 66 mole percent arabinose;

about 4.1 to about 4.5 mole percent rhamnose;

about 2.2 to about 2.4 mole percent xylose;

about 8.7 to about 9.7 mole percent galactouronic acid;

about 2.3 to about 2.5 mole percent mannose;

about 13.7 to about 15.1 mole percent galactose; and

about 4.2 to about 4.6 mole percent glucose,

as determined by combined gas chromatography/mass spectrometry (GC/MS)of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharidemethyl glycosides produced from the sample by acidic methanolysis. SeeExample 3.

Glycosyl linkage analysis was carried out by a modification of themethod of Hakomori, in which the polysaccharide composition wasdepolymerized, reduced, and acetylated, and the resultant partiallymethylated alditol acetates (PMAAs) analyzed by gas chromatography-massspectrometry (GC-MS) as described by York et al (1985) Methods Enzymol.118:3-40. This analysis revealed that, in particular embodiments, thepolysaccharide composition includes:

a terminally linked arabinofuranosyl residue (t-Araf);

a 2-linked arabinofuranosyl residue (2-Araf);

a 2-linked rhamnopyranosyl residue (2-Rhap);

a 3-linked arabinofuranosyl residue (3-Araf);

a terminally linked galactopyranosyl residue (t-Gal);

a 5-linked arabinofuranosyl residue (5-Araf);

a 3-linked glucopyranosyl residue (3-Glc) and/or a 2,4-linkedrhamnopyranosyl residue (2,4-Rhap);

a 2-linked glucopyranosyl residue (2-Glc);

a 4-linked mannopyranosyl residue (4-Man);

a 3,5-linked arabinofuranosyl residue (3,5-Araf);

a 2,5-linked arabinofuranosyl residue (2,5-Araf);

a 4-linked glucopyranosyl residue (4-Glc);

a 2,3,5-linked arabinofuranosyl residue (3,5-Araf) and/or a 2,3,4-linkedarabinopyranosyl residue (2,3,4-Arap);

a 4-linked galactouronic acid (4-gal A);

a 3,6-linked galactopyranosyl residue (3,6-Gal);

a 2,3,4,6-linked mannopyranosyl residue (2,3,4,6-Man);

a 2,3,4,6-linked galactopyranosyl residue (2,3,4,6-Gal)&2,3,4-linkedgalactouronic acid; and

a 2,3,4,6-linked glucopyranosyl residue (2,3,4,6-Glc).

In exemplary embodiments of the polysaccharide composition:

the terminally linked arabinofuranosyl residue (t-Araf) comprises about14.2 to about 15.7 wt percent of the polysaccharide;

the 2-linked arabinofuranosyl residue (2-Araf) comprises about 9.1 toabout 10.08 wt percent of the polysaccharide;

the 2-linked rhamnopyranosyl residue (2-Rhap) comprises about 0.3 wtpercent of the polysaccharide;

the 3-linked arabinofuranosyl residue (3-Araf) comprises about 3.0 toabout 3.4 wt percent of the polysaccharide;

the terminally linked galactopyranosyl residue (t-Gal) comprises about2.0 to about 2.2 wt percent of the polysaccharide;

the 5-linked arabinofuranosyl residue (5-Araf) comprises about 15.0 toabout 16.6 wt percent of the polysaccharide;

the 3-linked glucopyranosyl residue (3-Glc) and/or 2,4-linkedrhamnopyranosyl residue (2,4-Rhap) comprises about 0.7 wt percent of thepolysaccharide;

the 2-linked glucopyranosyl residue (2-Glc) comprises about 1.1 to about1.3 wt percent of the polysaccharide;

the 4-linked mannopyranosyl residue (4-Man) comprises about 1.3 to about1.5 wt percent of the polysaccharide;

the 3,5-linked arabinofuranosyl residue (3,5-Araf) comprises about 6.6to about 7.3 wt percent of the polysaccharide;

the 2,5-linked arabinofuranosyl residue (2,5-Araf) comprises about 5.0to about 5.6 wt percent of the polysaccharide;

the 4-linked glucopyranosyl residue (4-Glc) comprises about 4.6 to about5.0 wt percent of the polysaccharide;

the 2,3,5-linked arabinofuranosyl residue (3,5-Araf) and/or 2,3,4-linkedarabinopyranosyl residue (2,3,4-Arap) comprises about 25.7 to about 28.4wt percent of the polysaccharide;

the 4-linked galactouronic acid (4-gal A) comprises about 1.4 to about1.6 wt percent of the polysaccharide;

the 3,6-linked galactopyranosyl residue (3,6-Gal) comprises about 0.4 wtpercent of the polysaccharide;

the 2,3,4,6-linked mannopyranosyl residue (2,3,4,6-Man) comprises about0.7 wt percent of the polysaccharide;

the 2,3,4,6-linked galactopyranosyl residue (2,3,4,6-Gal)&2,3,4-linkedgalactouronic acid comprises about 2.0 to about 2.2 wt percent of thepolysaccharide; and

the 2,3,4,6-linked glucopyranosyl residue (2,3,4,6-Glc) comprises about2.1 to about 2.3 wt percent of the polysaccharide. See Example 4.

In certain embodiments, the polysaccharide composition is soluble inwater. In particular embodiments, the polysaccharide composition is verydrying on the tongue and tastes like chalk.

The polysaccharide composition of the invention binds to viruses andreduces the infectivity or pathogenicity of the virus. Without beingbound by any particular theory, the polysaccharide composition isbelieved to bind to a site that is conserved among viruses. Upon bindingto virus, a high-molecular weight component of the polysaccharidecomposition attaches to the virus, and a 150,000-180,000 molecularweight component (“150,000 molecular weight spin off component”) isreleased. In particular embodiments, the composition remains bound tovirus for at least 10 days, indicating that the binding may beirreversible. Compositions according to the invention can bindenveloped, non-enveloped, RNA, and DNA viruses, including, for example,Adenoviridae, Picornaviridae, Reoviridae, Arenaviridae, Bunyaviridae,Coroanviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae,Poxviridae Rhabdoviridae, Flaviviridae, and Retroviridae. See Example 6.

B. Preparation

The polysaccharide composition can be prepared from a suitable plantmaterial, such as, for example, material from one or more plants of theVitaceae family (e.g., grapes), the Ericaceae family (e.g., bilberriesand blueberries), the Fabaceae family (e.g., peanuts), and thePolygonaceae (e.g., Japanese knotweed).

In particular embodiments, the plant is a grape plant and the materialemployed is in the seeds, the skin, or a combination of the two. “Grapeseed extract” is commercially available from a variety of sources andcan be employed to prepare a substantially homogeneous aqueous mixtureor solution. For example, Spectrum Chemicals and Laboratory Products(Gardena, Calif.) sells grape seed extract percent powder G1273, whichcontain the seed and skin of Vitis vinifera. In accordance with certainembodiments, a 5 percent solution (25 gm/500 mL) of this powder in watercan be prepared as described in Example 1. The solution can be filteredusing a coarse filter paper (e.g., Whatman cat No 1213-185 18.5 cmcircle) to remove any large particles.

The homogeneous aqueous mixture or solution is then subjected to ionexchange and the colored product is recovered. In certain embodiments,the ion exchange resin employed is an anion exchange resin, such as aweak base anion exchange resin. For example, a Dowex™ M-43 resin, soldby Dow Chemical can be employed. This resin is Styrene-DVB macroporousresin with tertiary amine functional groups, and it has the typicalproperties shown in Table A1. The polysaccharide of the invention can beeluted from this column using distilled water.

TABLE A1 Properties of Dowex ™ M-43 Resin Typical Physical and ChemicalProperties FB (Free Base) Form Units Total Exchange Capacity, min. 1.55eq/L Weak Base Capacity, min. 1.35 eq/L Water Retention Capacity 40-50 %Particle Size Distribution Range  300-1200 μm >1200 μm, max. (16 mesh)<2 %  <300 μm, max. (50 mesh) <3 %

In particular embodiments, two ion exchange steps are carried out, e.g.,the colored product recovered from a first ion exchange column is passedover a second ion exchange column. The second ion exchange resin can bethe same as the first, for example the homogeneous aqueous mixture orsolution can be run through two sequential anion exchange columns, asdescribed in Example 1. In such embodiments, all of the colored eluatefrom the first column can be passed over a second, smaller column. In anexemplary embodiment, both columns include about 20 inches of resin, andthe first is about 3 inches in diameter, while the second is 1.5 inchesin diameter. A first fraction of the colored eluate from the secondcolumn can be discarded, for example, the first about 40 to about 60percent by volume (e.g., 50%). A second fraction of the colored eluateis collected, for example about 40 to about 60 percent by volume (e.g.,50%) or collection can be continued until the color of the eluatebecomes significantly lighter, e.g., almost clear. This second fractioncan then be further processed to obtain the polysaccharide compositionof the invention. In alternative embodiments, all of the colored eluateis collected from the second column and subjected to dialysis. SeeExample 1.

The colored product recovered after ion exchange can be concentrated,which can be carried out using any suitable concentration method, suchas dialysis or ultrafiltration. In various embodiments, the coloredproduct is concentrated by at least about 2-fold, at least about 5-fold,and at least about 10-fold.

In particular embodiments, the colored product recovered after ionexchange is further purified by removing low molecular weight material,e.g., material having a molecular weight of less than about 500,000Daltons, less than about 750,000 Daltons, or less than about 10⁶Daltons. Low molecular weight material can be removed by any suitablemeans, such as dialysis or ion trap. This removal step can be carriedout before or after concentration, or can be carried out by a singlestep that accomplishes both concentration and removal of low molecularweight material.

II. Pharmaceutical Formulations

The polysaccharide compositions of this invention can be formulated in apharmaceutically acceptable excipient, for example, for administrationto an individual in accordance with the methods of inhibiting viralinfectivity or pathogenicity or methods of inhibiting growth and/orproliferation of cancer cells, which are described below.Pharmaceutically acceptable excipients are described in Remington'sPharmaceutical Sciences (1980) 16th editions, Osol, ed., 1980.Pharmaceutically acceptable excipients can contain one or morephysiologically acceptable compound(s) that act, for example, tostabilize the composition or to increase or decrease the absorption ofthe polysaccharide composition. A pharmaceutically acceptable excipientsuitable for use in the invention is non-toxic to cells, tissues, orsubjects at the dosages employed, and can include a buffer (such as aphosphate buffer, citrate buffer, and buffers made from other organicacids), an antioxidant (e.g., ascorbic acid), a low-molecular weight(less than about 10 residues) peptide, a polypeptide (such as serumalbumin, gelatin, and an immunoglobulin), a hydrophilic polymer (such aspolyvinylpyrrolidone), an amino acid (such as glycine, glutamine,asparagine, arginine, and/or lysine), a monosaccharide, a disaccharide,and/or other carbohydrates (including glucose, mannose, and dextrins), achelating agent (e.g., ethylenediaminetetratacetic acid [EDTA]), a sugaralcohol (such as mannitol and sorbitol), a salt-forming counterion(e.g., sodium), and/or an anionic surfactant (such as Tween™,Pluronics™, and PEG). In one embodiment, the pharmaceutically acceptableexcipient is an aqueous pH-buffered solution.

Other pharmaceutically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable excipient(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the polysaccharide composition.

Pharmaceutical formulations of the invention can be stored in anystandard form, including, e.g., an aqueous solution or a lyophilizedcake. If appropriate, i.e., for injectables, such formulations aretypically sterile when administered to subjects. Sterilization of anaqueous solution is readily accomplished by filtration through a sterilefiltration membrane. If the formulation is stored in lyophilized form,the formulation can be filtered before or after lyophilization andreconstitution.

In particular embodiments, the polysaccharide composition is formulatedas a unit dosage formulation, i.e., a formulation that provides aspecific dose of polysaccharide composition. The unit dosage formulationprovides a therapeutically effective dose, or some fraction thereof,such that multiple doses can be taken to deliver a therapeuticallyeffective dose that is sufficiently well tolerated by the subject thatthe potential benefits of the dose outweigh the risks.

Exemplary dosage formulations include a powder, a solution, a tablet, acapsule, a lozenge, an ointment, a cream, a transdermal formulation(e.g., a patch), a gel, a nasal spray, a suppository, an injectable, animplantable sustained-release formulation, a lipid complex, etc. Incertain embodiments, one or more components of a solution can beprovided as a “concentrate,” e.g., in a storage container (e.g., in apremeasured volume) ready for dilution or in a soluble capsule ready foraddition to a volume of water.

In various embodiments, the polysaccharide compositions described hereincan be administered orally, in which case delivery can be enhanced bythe use of protective excipients. This is typically accomplished eitherby complexing the polysaccharide composition with a composition torender it resistant to acidic and enzymatic hydrolysis or by packagingthe agents in an appropriately resistant carrier, e.g. a liposome. Meansof protecting agents for oral delivery are well known in the art (see,e.g., U.S. Pat. No. 5,391,377).

Examplary formulations for topical delivery include, but are not limitedto, ointments and creams. Ointments are semisolid preparations which aretypically based on petrolatum or other petroleum derivatives. Creamscontaining the selected polysaccharide composition, are typicallyviscous liquid or semisolid emulsions, often either oil-in-water orwater-in-oil. Cream bases are typically water-washable and contain anoil phase, an emulsifier and an aqueous phase. The oil phase, alsosometimes called the “internal” phase, is generally comprised ofpetrolatum and a fatty alcohol such as cetyl or stearyl alcohol; theaqueous phase usually, although not necessarily, exceeds the oil phasein volume, and generally contains a humectant. The emulsifier in a creamformulation is generally a nonionic, anionic, cationic or amphotericsurfactant. The specific ointment or cream base to be used, as will beappreciated by those skilled in the art, is one that will provide foroptimum drug delivery. As with other carriers or vehicles, an ointmentbase is preferably inert, stable, nonirritating, and nonsensitizing.

III. Methods of Inhibiting Viral Infectivity and/or Pathogenicity

A. In General

Polysaccharide compositions according to the invention can be employedas an anti-viral agent against viruses that infect plant or animalcells. Accordingly, another aspect of the invention is a method ofinhibiting the infectivity and/or pathogenicity of a virus, whichentails contacting the virus with a polysaccharide composition of theinvention. As noted above, the polysaccharide composition binds andinhibits the infectivity of enveloped, non-enveloped, RNA, and DNAviruses, including, for example, Adenoviridae, Picornaviridae,Reoviridae, Arenaviridae, Bunyaviridae, Coroanviridae, Herpesviridae,Orthomyxoviridae, Paramyxoviridae, Poxviridae Rhabdoviridae,Flaviviridae, and Retroviridae.

The virus is generally contacted with polysaccharide composition underphysiological conditions (e.g., 37° C.), typically before or during aperiod of contact with cells that the virus is capable of infecting. Theduration of contact with the polysaccharide composition can vary,depending on the particular application of the method. The duration ofcontact can range from minutes to days or longer. For certainapplications, the polysaccharide composition is typically contacted withvirus for, e.g., about 10, about 20, about 30, about 40, or about 50mins.; or about 1, about 3, about 6, about 12, about 18, or about 24hours.

This method can be carried out in vitro, i.e., cell or tissue culture(see Example 6), or in vivo, i.e., in an organism. The cells/tissues canbe derived from, or the organism can be, a plant or animal, e.g., avertebrate, such as a bird or a mammal, and is preferably an animalhaving research or commercial value or value as pets, such as a mouse,rat, hamster, guinea pig, rabbit, cat, dog, chicken, pig, sheep, goat,cow, horse, as well as a monkey or other primate. In particularembodiments, human cells/tissues or a human subject is employed in themethod.

For in vivo applications, the composition can be administered to asubject who is already infected with a virus, to treat the infection(i.e., therapeutic use) or to a subject at risk for infection (i.e.,prophylactic use). In each case a route of administration is chosen soas to deliver the polysaccharide to a site containing, or likely tocontain, virus. For example, in the case of an influenza virus, thepolysaccharide composition can be administered as a nasal spray to anindividual who may be at risk for contracting an influenza infection(e.g., a healthcare worker). Alternatively, the polysaccharidecomposition can be used in a vaginal barrier cream to prevent cervicalherpes infections.

B. Administration

For in vitro applications, the polysaccharide composition can simply beadded to virus prior to, or during contact with cells.

For in vivo applications, the polysaccharide compositions identifiedherein are useful for local or systemic administration, for example,oral, sub-lingual, topical, injectable (including intrathecalinjection), systemic, nasal (or otherwise inhaled), rectal, or localadministration, such as by aerosol or transdermally, for prophylacticand/or therapeutic treatment of one or more of thepathologies/indications described herein.

Elevated serum half-life can be maintained by the use ofsustained-release “packaging” systems. Such sustained release systemsare well known to those of skill in the art (see, e.g., Tracy (1998)Biotechnol. Prog. 14: 108; Johnson et al. (1996), Nature Med. 2: 795;Herbert et al. (1998), Pharmaceut. Res. 15, 357).

In certain embodiments, the polysaccharide compositions may also bedelivered through the skin using conventional transdermal drug deliverysystems, i.e., transdermal “patches” wherein the polysaccharidecomposition is typically contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the drug composition is typically contained in a layer, or“reservoir,” underlying an upper backing layer. It will be appreciatedthat the term “reservoir” in this context refers to a quantity of“active ingredient(s)” that is ultimately available for delivery to thesurface of the skin. Thus, for example, the “reservoir” may include theactive ingredient(s) in an adhesive on a backing layer of the patch, orin any of a variety of different matrix formulations known to those ofskill in the art. The patch may contain a single reservoir, or it maycontain multiple reservoirs.

In one embodiment, the reservoir comprises a polymeric matrix of apharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the polysaccharide composition and any other materialsthat are present.

In certain embodiments, one or more polysaccharide compositionsdescribed herein are administered alone or in combination with one ormore other therapeutics in implantable (e.g., subcutaneous) matrices,termed “depot formulations.”

A major problem with standard drug dosing is that typical delivery ofdrugs results in a quick burst of medication at the time of dosing,followed by a rapid loss of the drug from the body. Most of the sideeffects of a drug occur during the burst phase of its release into thebloodstream. Secondly, the time the drug is in the bloodstream attherapeutic levels is very short; most is used and cleared during theshort burst.

Drugs (e.g., the polysaccharide compositions described herein) imbeddedin various matrix materials for sustained release can mitigate theseproblems. Drugs embedded, for example, in polymer beads or in polymerwafers have several advantages. First, most systems allow slow releaseof the drug, thus creating a continuous dosing of the body with smalllevels of drug. This typically prevents side effects associated withhigh burst levels of normal injected or pill-based drugs. Secondly,since these polymers can be made to release over hours to months, thetherapeutic span of the drug is markedly increased. Often, by mixingdifferent ratios of the same polymer components, polymers of differentdegradation rates can be made, allowing remarkable flexibility dependingon the polysaccharide composition being used. A long rate of drugrelease is beneficial for people who might have trouble staying onregular dosage, such as the elderly, but also represents an ease of useimprovement that everyone can appreciate. Most polymers can be made todegrade and be cleared by the body over time, so they will not remain inthe body after the therapeutic interval.

Another advantage of polymer-based drug delivery is that the polymersoften can stabilize or solubilize proteins, peptides, and other largemolecules that would otherwise be unusable as medications. Finally, manydrug/polymer mixes can be placed directly in the disease area, allowingspecific targeting of the medication where it is needed without losingdrug to the “first pass” effect. This is certainly effective fortreating the brain, which is often deprived of medicines that can'tpenetrate the blood/brain barrier.

A wide variety of approaches to designing depot formulations thatprovide sustained release of a polysaccharide composition are known andare suitable for use in the invention. Generally, the components of suchformulations are biocompatible and may be biodegradable. Biocompatiblepolymeric materials have been used extensively in therapeutic drugdelivery and medical implant applications to effect a localized andsustained release. See Leong et al., “Polymeric Controlled DrugDelivery,” Advanced Drug Delivery Rev., 1:199-233 (1987); Langer, “NewMethods of Drug Delivery,” Science, 249:1527-33 (1990); Chien et al.,Novel Drug Delivery Systems (1982). Such delivery systems offer thepotential of enhanced therapeutic efficacy and reduced overall toxicity.

Examples of classes of synthetic polymers that have been studied aspossible solid biodegradable materials include polyesters (Pitt et al.,“Biodegradable Drug Delivery Systems Based on Aliphatic Polyesters:Applications to Contraceptives and Narcotic Antagonists,” ControlledRelease of Bioactive Materials, 19-44 (Richard Baker ed., 1980);poly(amino acids) and pseudo-poly(amino acids) (Pulapura et al. “Trendsin the Development of Bioresorbable Polymers for Medical Applications,”J. Biomaterials Appl., 6:1, 216-50 (1992); polyurethanes (Bruin et al.,“Biodegradable Lysine Diisocyanate-based Poly(Glycolide-co-.epsilon.Caprolactone)-Urethane Network in Artificial Skin,” Biomaterials, 11:4,291-95 (1990); polyorthoesters (Heller et al., “Release of Norethindronefrom Poly(Ortho Esters),” Polymer Engineering Sci., 21:11, 727-31(1981); and polyanhydrides (Leong et al., “Polyanhydrides for ControlledRelease of Biopolysaccharide compositions,” Biomaterials 7:5, 364-71(1986).

Thus, for example, the polysaccharide composition can be incorporatedinto a biocompatible polymeric composition and formed into the desiredshape outside the body. This solid implant is then typically insertedinto the body of the subject through an incision. Alternatively, smalldiscrete particles composed of these polymeric compositions can beinjected into the body, e.g., using a syringe. In an exemplaryembodiment, the polysaccharide composition can be encapsulated inmicrospheres of poly (D,L-lactide) polymer suspended in a diluent ofwater, mannitol, carboxymethyl-cellulose, and polysorbate 80. Thepolylactide polymer is gradually metabolized to carbon dioxide andwater, releasing the polysaccharide composition into the system.

In yet another approach, depot formulations can be injected via syringeas a liquid polymeric composition. Liquid polymeric compositions usefulfor biodegradable controlled release drug delivery systems aredescribed, e.g., in U.S. Pat. Nos. 4,938,763; 5,702,716; 5,744,153;5,990,194; and 5,324,519. After injection in a liquid state or,alternatively, as a solution, the composition coagulates into a solid.

One type of polymeric composition suitable for this application includesa nonreactive thermoplastic polymer or copolymer dissolved in a bodyfluid-dispersible solvent. This polymeric solution is placed into thebody where the polymer congeals or precipitates and solidifies upon thedissipation or diffusion of the solvent into the surrounding bodytissues. See, e.g., Dunn et al., U.S. Pat. Nos. 5,278,201; 5,278,202;and 5,340,849 (disclosing a thermoplastic drug delivery system in whicha solid, linear-chain, biodegradable polymer or copolymer is dissolvedin a solvent to form a liquid solution).

The polysaccharide composition can also be adsorbed onto a membrane,such as a silastic membrane, which can be implanted, as described inInternational Publication No. WO 91/04014. Other exemplary implantablesustained release systems include, but are not limited to Re-Gel®,SQ2Gel®, and Oligosphere® by MacroMed, ProLease® and Medisorb® byAlkermes, Paclimer® and Gliadel® Wafer by Guilford pharmaceuticals, theDuros implant by Alza, acoustic biSpheres by Point Biomedical, theIntelsite capsule by Scintipharma, Inc., and the like.

The polysaccharide composition can be co-administered with one or moreadditional agents that inhibit viral infectivity and/or pathogenicity bysimultaneous administration or sequential administration.

Additional agents can be administered by a route that is the same as, ordifferent from, the route of administration of the polysaccharidecomposition. Where possible, it is generally desirable to administerthese agents by the same route of administration, preferably in the sameformulation. However, differences in pharmacodynamics, pharmacokinetics,or other considerations may dictate the co-administration of thepolysaccharide composition and additional agent(s) in separateformulations. Additional agents can be administered according tostandard practice.

C. Dose

In particular embodiments, virus are contacted in vitro with an amountof polysaccharide composition effective to reduce infection of cells byabout 25 percent, about 50 percent, about 75 percent, about 80 percent,about 90 percent, about 95 percent, about 96 percent, about 97 percent,about 98 percent, about 99 percent, or about 100 percent.

In in vivo applications, the compositions of this invention areadministered, for example, to a subject in an amount sufficient (onaverage) to inhibit, by any of the percentages listed above, or preventinfectivity and/or pathogenicity of the virus. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend upon the severity of thecondition and the general state of the subject's health. Single ormultiple administrations of the compositions may be administereddepending on the dosage and frequency as required and tolerated by thesubject.

The concentration of polysaccharide composition can vary widely and willbe selected primarily based on fluid volumes, viscosities, body weightand the like in accordance with the particular mode of administrationselected and the subject's needs. In accordance with standard practice,the clinician can titer the dosage and modify the route ofadministration as required to obtain the optimal therapeutic effect.Generally, the clinician begins with a low dose and increases the dosageuntil the desired therapeutic effect is achieved. Starting doses for agiven active agent can, for example be extrapolated from in vitro and/oranimal data.

In particular embodiments, concentrations of active agent(s) willtypically be selected to provide dosages ranging from about 0.1 or 1mg/kg/day to about 300 mg/kg/day and sometimes higher. Typical dosagesfor an inhalant formulation of the polysaccharide composition can beabout 1, about 3, about 5, about 10, about 15, about 20, about 25, about30, about 35, about 40, about 45, about 50, about 55, about 60, about 65and about 70 mg/kg/day, or any range having any of these values as anendpoint. Polysaccharide composition formulations can be administered inone or multiples doses a day. In some cases, one administration may besufficient to achieve the desired results. Alternatively, polysaccharidecomposition can be administered over multiples days as needed. Incertain embodiments, polysaccharide compositions of the invention can beadministered in an intravenous (IV) drip at a dosage of about 10, about15, about 20, about 25, about 30, about 35, about 40, about 45, about50, about 75, about 100, about 125, about 150 about 175, about 200,about 250, about 300, and about 350 mg/kg/hour, or any range having anyof these values as an endpoint. Treatment can be conducted, for example,over about 1 to about 8 hours, e.g., about 4 hours. It will beappreciated that such dosages may be varied to optimize a therapeuticregimen in a particular subject or group of subjects, and thus any ofthese values can represent the upper or lower limit of a suitable dosagerange according to the invention.

In embodiments of the method in which an additional agent that inhibitsviral infectivity and/or pathogenicity is co-administered with thepolysaccharide composition, suitable doses of additional agents areknown and can be adjusted by the clinician for co-administration withthe polysaccharide composition.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

IV. Methods of Inhibiting Growth and/or Proliferation of Cancer Cells

The invention also provides a method of inhibiting the growth and/orproliferation of a cancer cell. The method entails contacting the cellwith a polysaccharide composition according to the invention. As usedherein, the phrase “contacting the cell with a polysaccharidecomposition according to the invention” includes introducing thiscomposition into the cellular environment such that the composition isin the proximity of the target cells.

The cell is generally contacted with polysaccharide composition underphysiological conditions. The duration of contact with thepolysaccharide composition can vary, depending on the particularapplication of the method. The duration of contact can range fromminutes to days or longer. For certain applications, the polysaccharidecomposition is typically contacted with the cell for, e.g., about 30mins.; or about 1, about 3, about 6, about 12, or about 24 hours. Tokill cancer cells, the duration of contact is typically at least about 3hours.

This method can be carried out in vitro, i.e., cell or tissue culture(see Example 8), or in vivo, i.e., in an organism. The cells/tissues canbe derived from, or the organism can be an organism that is susceptibleto cancer, including any of those described above with respect to viralinhibition. In particular embodiments, mammalian, particularly human,cells/tissues or a mammalian, particularly human, subject is employed inthe method. The cell can be any type of cancer cell, i.e., any cell thatdisplays a transformed phenotype (e.g., growth in soft agar) and/oruncontrolled proliferation. In particular embodiments, the cancer cellis a non-solid cancer cell (e.g., a leukemia or lymphoma cell). Inparticular embodiments, the cancer cell is a solid tumor cell. Incertain embodiments, the cancer cell is a metastatic cancer cell. Anycancer cell can be inhibited by the polysaccharide composition of theinvention, including bladder cancer, breast cancer, colon and/or rectalcancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lungcancer, melanoma, non-Hodgkin lymphoma, ovarian cancer, pancreaticcancer, prostate cancer, skin cancer (non-melanoma), stomach cancer, andthyroid cancer.

For in vitro applications, the polysaccharide composition can simply beadded to cells. The routes and considerations for in vivo administrationare essentially as described above with respect to viral inhibition.

In particular embodiments, cancer cells are contacted in vitro in withan amount of polysaccharide composition effective to reduce growthand/or proliferation of cancer cells by about 50 percent, about 75percent, about 80 percent, about 90 percent, about 95 percent, about 96percent, about 97 percent, about 98 percent, about 99 percent, or about100 percent. In preferred embodiments, the amount of polysaccharidecomposition employed is sufficient to kill the cancer cells.

In in vivo applications, the compositions of this invention areadministered, for example, to a subject in an amount sufficient (onaverage) to inhibit cancer cell growth and/or proliferation, by any ofthe percentages listed above or to eradicate the cancer. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.” Amounts effective for this use will depend upon the severity ofthe condition and the general state of the subject's health. Single ormultiple administrations of the composition may be administereddepending on the dosage and frequency as required and tolerated by thesubject. General considerations for determining a suitable dosingregimen the polysaccharide composition are essentially as describedabove for viral inhibition.

In certain embodiments, an additional chemotherapeutic agent isco-administered with the polysaccharide composition, simultaneously orsequentially. Suitable doses of additional agents are known and can beadjusted by the clinician for co-administration with the polysaccharidecomposition.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

V. Methods of Treating Pain and/or Inflammation

The invention also includes a method of using the polysaccharidecomposition of the invention to treat pain and/or inflammation. Forexample, the composition can be used to treat diseases characterized bya “hyperactivity” of the immune system and/or where the production ofnatural virus-like structures causes disease; examples includeautoimmune diseases, e.g., rheumatoid arthritis, or other chronicinflammatory diseases. The method entails administering to a subject inneed thereof, an effective amount of a polysaccharide compositionaccording to the invention.

The subject of the method can be any organism in which it is desirableto treat pain and/or inflammation, including any of those describedabove with respect to viral inhibition. In particular embodiments, amammalian, particularly a human, subject is employed in the method. Inparticular embodiments, the polysaccharide is applied topically toreduce pain experienced at or near the surface of the body.

In one embodiment, the subject suffers from inflammatory pain, which maybe acute or chronic. A polysaccharide composition can be administered toreduce pain and/or inflammation. Examples of inflammation amenable totreatment by administering a polysaccharide composition of the inventioninclude pain/inflammation due to trauma, such as cuts, stings (e.g., beeor wasp stings) or other reactions to venom (e.g., jellyfish), burn,sunburn; dermatitis; neuritis; mucositis; urethritis; cystitis;gastritis; pneumonitis; and collagen vascular disease.

In another embodiment, the subject suffers from neuropathic pain, whichalso may be acute or chronic. Examples of neuropathic pain amenable totreatment by administering a polysaccharide composition of the inventioninclude pain due to conditions such as, e.g., neuralgia, causalgia(complex regional pain syndrome type II), diabetes, collagen vasculardisease, trigeminal neuralgia, spinal cord injury, brain stem injury,thalamic pain syndrome, complex regional pain syndrome type I/reflexsympathetic dystrophy, Fabry's syndrome, small fiber neuropathy, cancer,cancer chemotherapy, chronic alcoholism, stroke, abscess, demyelinatingdisease, viral infection, anti-viral therapy, AIDS, AIDS therapy,post-polio syndrome, and post-herpetic neuralgia. Neuropathic painarising from, e.g., trauma, surgery, amputation, toxin, and/orchemotherapy can also be treated using the antagonists of the invention.

In particular embodiments, the subject suffers from a generalized paindisorder, such as, e.g., fibromyalgia, irritable bowel syndrome, and/ortemporomandibular disorders.

Other indications for the method of the invention include allodynia,migraine, atypical facial pain, back pain, arthritic pain, sports injurypain, pain related to infection (e.g. HIV), labor pain, post-operativepain, conditions associated with visceral pain, angina, and urinarybladder incontinence (e.g. urge incontinence).

The routes and considerations for administration are essentially asdescribed above with respect to viral inhibition. For topicaladministration, any suitable topical formulation (e.g., as describedabove) containing a polysaccharide of the invention is simply applied tothe skin over the region affected by the pain and/or inflammation. Thepolysaccharide composition is present in the topical formulation in anamount sufficient to reduce pain and/or inflammation. The number ofapplications per day will depend upon a number of factors, including thetype of formulation employed and the activities of the subject afterapplication (i.e., if the subject must wash the area frequently, thepolysaccharide composition may need to be reapplied more frequently.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

VI. Immunogenic Compositions and Methods of Inducing an Immune Response

In other embodiments, the invention provides an immunogenic composition,said which includes a polysaccharide composition of the invention boundto a virus. The polysaccharide composition can be bound to the virus asdescribed above with respect to viral inhibition. Suitable virusesinclude any to which the polysaccharide composition binds, e.g.,enveloped, non-enveloped, RNA, and DNA viruses, including, for example,Adenoviridae, Picornaviridae, Reoviridae, Arenaviridae, Bunyaviridae,Coroanviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae,Poxviridae Rhabdoviridae, Flaviviridae, and Retroviridae.

The viral-polysaccharide complex is employed at the desired degree ofpurity and at a sufficient concentration to induce an immune responseand is typically mixed with a pharmaceutically acceptable excipient. Inparticular embodiments, the immunogenic composition is formulated forinjection, e.g., intramuscular, intradermal, or subcutaneous injection,although other routes of administration, including oral and intranasal,are contemplated as well. Suitable carriers for injection includesterile water, but preferably are physiologic salt solutions, such asnormal saline or buffered salt solutions (e.g., phosphate-bufferedsaline or ringer's lactate).

Immunogenic compositions according to the invention contain an adjuvant,although this is not essential. Useful adjuvants include QS21 (Quillajasaponaria, commercially available from Cambridge Biotech, Worcester,Mass.), which stimulates cytotoxic T-cells, and alum (aluminum hydroxideadjuvant). Formulations with different adjuvants that enhance cellularor local immunity can also be used. In particular, immunopotentiatorssuch as cytokines can be included in the immunogenic composition.Examples of suitable immunopotentiating cytokines include interleukins,such as interleukin-2 (IL-2) and interleukin-12 (IL-12), and tumornecrosis factor-alpha (TNF-α).

Additional excipients that can be present in the immunogenic compositioninclude low molecular weight polypeptides (less than about 10 residues),proteins, amino acids, carbohydrates including glucose or dextrans,chelating agents such as EDTA, and other excipients that stabilize thepolysaccharide composition and/or the virus and/or the complex orinhibit growth of microorganisms.

Preferably, any virus in the immunogenic composition has beeninactivated, for example, by binding to the polysaccharide compositionof the invention, as described above with respect to viral inhibition.Suitable relative amounts of polysaccharide composition and virus toachieve viral inactivation can be determined from Examples 5 and 7, orempirically, by carrying out studies such as those described in theseexamples.

Exemplary immunogenic compositions according to the invention caninclude virus in a range of approximately 10⁶TCID₅₀ to approximately10³TCID₅₀ depending on the route of administration of virus. (“TCID”refers to the “tissue culture infective dose,” which is that amount of apathogenic agent that will produce pathological change when inoculatedon tissue cultures; TCID₅₀, refers to the amount that produces apathological change in 50% of all cell cultures). Adjuvants or othercomponents are used at concentrations in accordance with standardpractice or at concentrations empirically determined to provide thedesired effect(s).

The amount of immunogenic composition administered is generally onesufficient to elicit a measurable immune response, such as, e.g., aneutralizing antibody response, increase in interferon, increase innatural killer cell activity and/or survival of a lethal infection.Depending on formulation and route of administration, such response(s)can be achieved after one immunization or after one or more boosterimmunizations, following an initial immunization. Where the immuneresponse is an antibody response, neutralizing or otherwise, antibodiesinduced by the immunogenic composition can be used in a diagnostic todetect the virus strain used in the immunogen or to affinity-purify suchvirus or to inactivate such virus.

In particular embodiments, the dosing regimen is sufficient to elicit anin vivo protective immune response, i.e., one in which an organism isprotected from homologous viral challenge (i.e., challenge with the sameviral strain as that included in the immunogenic composition). Anyanimal that mounts an immune response to the viral-polysaccharidecomposition-viral complex immunogen can be the subject of animmunization method according to the invention, including any of thosedescribed above with respect to viral inhibition. In particularembodiments, mammalian, particularly human, subjects are employed in themethod. Suitable subjects can be seronegative or seropositive for thevirus strain included in the immunogen. In the case of seronegativesubjects, the dosing regimen will generally be designed to elicit aprotective immune response, such that the risk of infection uponsubsequent exposure to the virus is reduced by about 25 percent, about50 percent, about 75 percent, about 80 percent, about 90 percent, about95 percent, about 96 percent, about 97 percent, about 98 percent, about99 percent, about 100 percent, or by a range having endpoints defined byany of the above values. In the case of seropositive subjects, thedosing regimen will generally be designed to reduce a measure ofinfection, such as, e.g., viral load, by about these same percentages.

In preferred embodiments, the dosing regimen is sufficient to elicit anin vivo protective immune response to heterologous viral challenge(i.e., challenge with a different strain of the type of virus includedin the immunogenic composition). The regimen may vary depending on thevirus, and the determination of a suitable regimen can be determined byone of skill in the art. In some cases, it may be advantageous to boost,e.g., annually or biannually after protection has be achieved. Inaddition, one or more immune responses, e.g., neutralizing antibodylevels, can be assayed and the regimen adjusted accordingly.

VII. Methods of Ameliorating Symptoms of Aging

Another aspect of the invention is a method of ameliorating a symptom ofaging in a subject in need thereof. The method entails administering tothe subject an effective amount of a polysaccharide compositionaccording to the invention, wherein the effective amount is sufficientto ameliorate a least one symptom of aging.

The subject of the method can be any organism in which it is desirableto treat pain and/or inflammation, including any of those describedabove with respect to viral inhibition. In particular embodiments, amammalian, particularly a human, subject is employed in the method.Illustrative symptoms of aging that can be ameliorated by this methodinclude, but are not limited to, hair loss, near vision loss due toaging, age spots, skin thinning and/or loss of elasticity, reducedhormone levels due to aging, loss of stamina due to aging, increased fatdeposits, chronic joint and back pains, and/or age-related impairmentsin cognition.

The routes and considerations for administration are essentially asdescribed above with respect to viral inhibition. The number of dosesper day will depend upon a number of factors, including the type offormulation employed and the route of administration.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

VIII. Methods and Devices for Detecting/Quantifying Virus

The invention also provides a method of detecting and/or quantifying avirus. This method entails contacting a sample with a polysaccharidecomposition of the invention and detecting and/or quantifying binding tosaid composition. This method is useful for assessing a sample for thepresence and/or amount of a virus and can be employed in identifying avirus.

A. Sample Collection and Processing

The assay methods of the invention can be carried out on any samplesuspected of containing a virus, but are generally carried out onbiological samples derived from a plant or animal (including any ofthose described herein), preferably a mammal, and more preferably ahuman. Convenient samples include, for example, tissue, blood, serum,plasma, urine, and saliva.

The sample may be pretreated as necessary by dilution in an appropriatebuffer solution or concentrated, if desired. Any of a number of standardaqueous buffer solutions, employing any of a variety of buffers, such asphosphate, Tris, or the like, at physiological pH, can be used.

B. Assaying for Virus

Virus can be detected and quantified by assaying binding to thepolysaccharide composition using any of a number of binding assayformats well known to those of skill in the art.

In particular embodiments, virus is detected and/or quantified in thebiological sample using any of the available immunoassay formats, inwhich the anti-analyte antibody is replaced by the polysaccharidecomposition of the invention (see, e.g., U.S. Pat. Nos. 4,366,241;4,376,110; 4,517,288; and 4,837,168). For a general review ofimmunoassays, see also Methods in Cell Biology Volume 37: Antibodies inCell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic andClinical Immunology 7th Edition, Stites & Terr, eds. (1991).

Conventional immunoassays often utilize a “capture agent” tospecifically bind to and often immobilize the analyte on a solid phase.Thus, the polysaccharide composition of the invention can be employed asa capture agent for a virus of interest (e.g., any of those describedhere). Polysaccharide compositions according to the invention can beaffixed to a solid phase via any available means for immobilizingpolysaccharides, such as the 2-amino-methyl N,O-hydroxyethyl spacerdescribed by Bohorov, O. et al. (2006) Glycobiology 16(12):21C-27C,which is hereby incorporated by reference in its entirety; see alsoKamitani, R. et al. (2007) Bulletin of the Chemical Society of Japan80(9):1808-1813 (describing the attachment of carbohydrates to polymersurfaces), which is hereby incorporated by reference in its entirety;U.S. Pat. No. 7,241,453, issued to Engel et al. on Jul. 10, 2007(discussing the production of anti-microbial surfaces through theattachment of carbohydrates), which is hereby incorporated by referencein its entirety; Weiping, Q et al. (1999) J. Inclusion Phenomena andMacrocyclic Chemistry 35(1-2) (describing the attachment of oxidizedcarbohydrates to other moieties), which is hereby incorporated byreference in its entirety; corninfo.ps.uci.edu/writings/surfacechem.html(describing the non-covalent attachment of carbohydrates to metalsurfaces); Moritz, B. B. et al. (2005) ChemBioChem 6(6): 1007-1015)(describing the attachment of carbohydrates to glass surfaces), which ishereby incorporated by reference in its entirety;microarrays.ca/support/emerg_tech_docs/CarbArrays_July2007.pdf(describing the attachment of carbohydrates to glass surfaces); Lee,J-C. et al. (2006) Angewandte Chemie International Ed. 45:2753-57(describing the use of a photocleavable linker to reversibly attachcarbohydrates to polymer surfaces), which is hereby incorporated byreference in its entirety.

Immunoassays also typically utilize a labeled detection agent tospecifically bind to and label the binding complex formed by the captureagent and the analyte. The labeled detection agent may itself be one ofthe moieties making up the antibody/analyte complex. Alternatively, thelabeled detection agent may be a third moiety, such as another antibody,that specifically binds to the capture agent/analyte complex. Otherpolypeptides capable of specifically binding immunoglobulin constantregions, such as polypeptide A or polypeptide G may also make up thelabeled detection agent. These polypeptides are normal constituents ofthe cell walls of streptococcal bacteria. They exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, generally Kronval, et al. (1973) J. Immunol.,111: 1401-1406, and Akerstrom (1985) J. Immunol., 135: 2589-2542). Theuse of an anti-viral antibody as the labeled detection agent will aid inthe identification and/or quantification of specific viruses. In otherembodiments, the labeled detection agent can be a labeled form of thepolysaccharide composition of the invention. Such embodiments wouldpermit the detection and/or quantification of virus, but not itsidentification, since the polysaccharide composition does notdistinguish between different types of virus.

Preferred immunoassays for detecting the target polypeptide(s) areeither competitive or noncompetitive. Noncompetitive immunoassays areassays in which the amount of captured analyte is directly measured. Incompetitive assays, the amount of analyte in the sample is measuredindirectly by measuring the amount of an added (exogenous) labeledanalyte displaced (or competed away) from a capture agent by the analytepresent in the sample. In one competitive assay, a known amount of, inthis case, labeled virus is added to the sample, and the sample is thencontacted with a capture agent. The amount of labeled virus bound to theantibody is inversely proportional to the concentration of virus presentin the sample.

The assays of this invention are scored (as positive or negative orquantity of analyte) according to standard methods well known to thoseof skill in the art. The particular method of scoring will depend on theassay format and choice of label. For example, an assay can be scored byvisualizing the colored product produced by the enzymatic label. Aclearly visible colored band or spot at the correct molecular weight isscored as a positive result, while the absence of a clearly visible spotor band is scored as a negative. The intensity of the band or spot canprovide a quantitative measure of analyte concentration.

C. Solid Phase

For embodiments of the invention that employ a solid phase as a supportfor the capture agent, the solid phase can be any suitable porousmaterial with sufficient porosity to allow access by reagents and asuitable surface affinity to bind a capture agent. Microporousstructures are generally preferred, but materials with gel structure inthe hydrated state may be used as well. Useful solid supports include:natural polymeric carbohydrates and their synthetically modified,crosslinked, or substituted derivatives, such as agar, agarose,cross-linked alginic acid, substituted and cross-linked guar gums,cellulose esters, especially with nitric acid and carboxylic acids,mixed cellulose esters, and cellulose ethers; natural polymerscontaining nitrogen, such as proteins and derivatives, includingcross-linked or modified gelatins; natural hydrocarbon polymers, such aslatex and rubber; synthetic polymers which may be prepared with suitablyporous structures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel,quartz, or glass (these materials may be used as filters with the abovepolymeric materials); and mixtures or copolymers of the above classes,such as graft copolymers obtained by initializing polymerization ofsynthetic polymers on a pre-existing natural polymer. All of thesematerials may be used in suitable shapes, such as a planar substrate,e.g., films, sheets, or plates, or they may be coated onto, bonded, orlaminated to appropriate inert carriers, such as paper, glass, plasticfilms, fabrics, or the like.

The porous structure of nitrocellulose has excellent absorption andadsorption qualities for a wide variety of reagents including monoclonalantibodies. Nylon also possesses similar characteristics and also issuitable.

Porous solid phases useful in the invention can be in the form of sheetsof thickness from about 0.01 to 0.5 mm, e.g., about 0.1 mm. The poresize may vary within wide limits, and is preferably from about 0.025 toabout 15 microns, especially from about 0.15 to about 15 microns.

Preferred solid phase materials for flow-through assay devices includefilter paper such as a porous fiberglass material or other fiber matrixmaterials. The thickness of such material is not critical and will be amatter of choice, largely based upon the properties of the sample oranalyte being assayed, such as the fluidity of the biological sample.

Alternatively, the solid phase can constitute a bead, e.g., amicroparticle. Microparticles useful in the invention can be selected byone skilled in the art from any suitable type of particulate materialand include those composed of polystyrene, polymethylacrylate,polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile,polycarbonate, or similar materials.

Microparticles can be suspended in the mixture of soluble reagents andbiological sample or can be retained and immobilized by a supportmaterial. In the latter case, the microparticles on or in the supportmaterial are not capable of substantial movement to positions elsewherewithin the support material.

The methods of the present invention can be adapted for use in systemsthat utilize microparticle technology including automated andsemi-automated systems wherein the solid phase comprises amicroparticle. Such systems include those described in pending U.S. App.No. 425,651 and U.S. Pat. No. 5,089,424, which correspond to publishedEPO App. Nos. EP 0 425 633 and EP 0 424 634, respectively, and U.S. Pat.No. 5,006,309.

In particular embodiments, the solid phase includes one or moreelectrodes. Capture agent(s) can be affixed, directly or indirectly, tothe electrode(s). In one embodiment, for example, capture agents can beaffixed to magnetic or paramagnetic microparticles, which are thenpositioned in the vicinity of the electrode surface using a magnet.Systems in which one or more electrodes serve as the solid phase areuseful where detection is based on electrochemical interactions.Exemplary systems of this type are described, for example, in U.S. Pat.No. 6,887,714 (issued May 3, 2005). Other possible supports for assay ofthe invention include capillaries, microchannels, and syringes, etc.

The capture agent can be attached to the solid phase by adsorption onthe porous material, where it is retained by hydrophobic forces.Alternatively, the surface of the solid phase can be activated bychemical processes that cause covalent linkage of the capture agent tothe support.

To change or enhance the intrinsic charge of the solid phase, a chargedsubstance can be coated directly onto the solid phase material or ontomicroparticles which then are retained by a solid phase material. Ioncapture procedures for immobilizing an immobilizable reaction complexwith a negatively charged polymer, described in U.S. App. No. 150,278,corresponding to EP Publication No. 0326100, and U.S.App. No. 375,029(EP Publication No. 0406473), can be employed according to the presentinvention to affect a fast solution-phase immunochemical reaction. Inthese procedures, an immobilizable reaction complex is separated fromthe rest of the reaction mixture by ionic interactions between thenegatively charged polyanion/immune complex and the previously treated,positively charged porous matrix and detected by using any of a numberof signal-generating systems, including, e.g., chemiluminescent systems,as described in U.S. App. No. 921,979, corresponding to EPO PublicationNo. 0 273,115.

If the solid phase is silicon or glass, the surface must generally beactivated prior to attaching the specific binding partner. Activatedsilane compounds such as triethoxy amino propyl silane (available fromSigma Chemical Co., St. Louis, Mo.), triethoxy vinyl silane (AldrichChemical Co., Milwaukee, Wis.), and (3-mercapto-propyl)-trimethoxysilane (Sigma Chemical Co., St. Louis, Mo.) can be used to introducereactive groups such as amino-, vinyl, and thiol, respectively. Suchactivated surfaces can be used to link the capture directly (in thecases of amino or thiol), or the activated surface can be furtherreacted with linkers such as glutaraldehyde, bis (succinimidyl)suberate, SPPD 9 succinimidyl 3-[2-pyridyldithio] propionate), SMCC(succinimidyl-4-[Nmaleimidomethyl] cyclohexane-1-carboxylate), SIAB(succinimidyl [4iodoacetyl] aminobenzoate), and SMPB (succinimidyl4-[1maleimidophenyl] butyrate) to separate the capture agent from thesurface. Vinyl groups can be oxidized to provide a means for covalentattachment. Vinyl groups can also be used as an anchor for thepolymerization of various polymers such as poly-acrylic acid, which canprovide multiple attachment points for specific capture agents. Aminogroups can be reacted with oxidized dextrans of various molecularweights to provide hydrophilic linkers of different size and capacity.Examples of oxidizable dextrans include Dextran T-40 (molecular weight40,000 daltons), Dextran T-110 (molecular weight 110,000 daltons),Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M(molecular weight 2,000,000 daltons) (all of which are available fromPharmacia, Piscataway, N.J.), or Ficoll (molecular weight 70,000daltons; available from Sigma Chemical Co., St. Louis, Mo.).Additionally, polyelectrolyte interactions can be used to immobilize aspecific capture agent on a solid phase using techniques and chemistriesdescribed U.S. App. No. 150,278, filed Jan. 29, 1988, and U.S. App. No.375,029, filed Jul. 7, 1989, each of which is incorporated herein byreference.

Other considerations affecting the choice of solid phase include theability to minimize non-specific binding of labeled entities andcompatability with the labeling system employed. For, example, solidphases used with fluorescent labels should have sufficiently lowbackground fluorescence to allow signal detection.

Following attachment of a specific capture agent, the surface of thesolid support may be further treated with materials such as serum,proteins, or other blocking agents to minimize non-specific binding.

D. Labeling Systems

As discussed above, many assays according to the invention employ alabeled detection agent.

Detectable labels suitable for use in the detection agents of thepresent invention include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical, orchemical means. Useful labels in the present invention include magneticbeads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, texasred, rhodamine, green fluorescent protein, and the like, see, e.g.,Molecular Probes, Eugene, Oreg., USA), chemiluminescent compounds suchas acridinium (e.g., acridinium-9-carboxamide), phenanthridinium,dioxetanes, luminol and the like, radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C,or ³²P), catalysts such as enzymes (e.g., horse radish peroxidase,alkaline phosphatase, beta-galactosidase and others commonly used in anELISA), and colorimetric labels such as colloidal gold (e.g., goldparticles in the 40-80 nm diameter size range scatter green light withhigh efficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

The label can be attached to the detection agent prior to, or during, orafter contact with the biological sample. So-called “direct labels” aredetectable labels that are directly attached to or incorporated intodetection agents prior to use in the assay. Direct labels can beattached to or incorporated into detection agents by any of a number ofmeans well known to those of skill in the art.

In contrast, so-called “indirect labels” typically bind to the detectionagent at some point during the assay. Often, the indirect label binds toa moiety that is attached to or incorporated into the detection agentprior to use. Thus, for example, an antibody used as a detection agent(a “detection antibody”) can be biotinylated before use in an assay.During the assay, an avidin-conjugated fluorophore can bind thebiotin-bearing detection agent, to provide a label that is easilydetected.

In another example of indirect labeling, polypeptides capable ofspecifically binding immunoglobulin constant regions, such aspolypeptide A or polypeptide G, can also be used as labels for detectionantibodies. Such polypeptides can thus be labeled and added to the assaymixture, where they will bind to the detection antibody.

Some labels useful in the invention may require the use of an indicatorreagent to produce a detectable signal. In an ELISA, for example, anenzyme label (e.g., beta-galactosidase) will require the addition of asubstrate (e.g., X-gal) to produce a detectable signal.

E. Formats

The assays of the invention can be conducted using any format known inthe art.

1. Fluorescence Polarization Immunoassay (FPIA)

In an exemplary embodiment, a fluorescent label is employed in afluorescence polarization immunoassay (FPIA) according to the invention.Generally, fluorescent polarization techniques are based on theprinciple that a fluorescent label, when excited by plane-polarizedlight of a characteristic wavelength, will emit light at anothercharacteristic wavelength (i.e., fluorescence) that retains a degree ofthe polarization relative to the incident light that is inverselyrelated to the rate of rotation of the label in a given medium. As aconsequence of this property, a label with constrained rotation, such asone bound to another solution component with a relatively lower rate ofrotation, will retain a relatively greater degree of polarization ofemitted light than when free in solution.

This technique can be employed in assays according to the invention, forexample, by selecting reagents such that binding of the fluorescentlylabeled entities forms a complex sufficiently different in size suchthat a change in the intensity light emitted in a given plane can bedetected.

Fluorophores useful in FPIA include fluorescein, aminofluorescein,carboxyfluorescein, and the like, preferably 5 and6-aminomethylfluorescein, 5 and 6-aminofluorescein,6-carboxyfluorescein, 5-carboxyfluorescein, thioureafluorescein, andmethoxytriazinolyl-aminofluorescein, and similar fluorescentderivatives. Examples of commercially available automated instrumentswith which fluorescence polarization assays can be conducted include:IMx® system, TDx® system, and TDxFLx™ system (all available from AbbottLaboratories, Abbott Park, Ill.).

2. Scanning Probe Microscopy (SPM)

The use of scanning probe microscopy (SPM) for assays also is atechnology to which the immunoassay methods of the present invention areeasily adaptable. In SPM, in particular, in atomic force microscopy, thecapture agent is affixed to a solid phase having a surface suitable forscanning. The capture agent can, for example, be adsorbed to a plasticor metal surface. Alternatively, the capture agent can be covalentlyattached to, e.g., derivatized plastic, metal, silicon, or glassaccording to methods known to those of ordinary skill in the art.Following attachment of the capture agent, the biological sample iscontacted with the solid phase, and a scanning probe microscope is usedto detect and quantify solid phase-affixed complexes. The use of SPMeliminates the need for labels which are typically employed inimmunoassay systems. Such a system is described in U.S. App. No.662,147, which is incorporated herein by reference.

3. MicroElectroMechanical Systems (MEMS)

Assays according to the invention can also be carried out using aMicroElectroMechanical System (MEMS). MEMS are microscopic structuresintegrated onto silicon that combine mechanical, optical, and fluidicelements with electronics, allowing convenient detection of an analyteof interest. An exemplary MEMS device suitable for use in the inventionis the Protiveris' multicantilever array. This array is based onchemo-mechanical actuation of specially designed siliconmicrocantilevers and subsequent optical detection of the microcantileverdeflections. When coated on one side with a binding partner, amicrocantilever will bend when it is exposed to a solution containingthe complementary molecule. This bending is caused by the change in thesurface energy due to the binding event. Optical detection of the degreeof bending (deflection) allows measurement of the amount ofcomplementary molecule bound to the microcantilever.

4. Electrochemical Detection Systems

In other embodiments, assays according to the invention are carried outusing electrochemical detection. A basic procedure for electrochemicaldetection has been described by Heineman and coworkers. This entailedimmobilization of a primary antibody (Ab, rat-anti mouse IgG), followedby exposure to a sequence of solutions containing the antigen (Ag, mouseIgG), the secondary antibody conjugated to an enzyme label (AP-Ab, ratanti mouse IgG and alkaline phosphatase), and p-aminophenyl phosphate(PAPP). The AP converts PAPP to p-aminophenol (PAP_(R), the “R” isintended to distinguish the reduced form from the oxidized form,PAP_(O), the quinoneimine), which is electrochemically reversible atpotentials that do not interfere with reduction of oxygen and water atpH 9.0, where AP exhibits optimum activity. PAP_(R) does not causeelectrode fouling, unlike phenol whose precursor, phenylphosphate, isoften used as the enzyme substrate. Although PAP_(R) undergoes air andlight oxidation, these are easily prevented on small scales and shorttime frames. Picomole detection limits for PAP_(R) and femtogramdetection limits for IgG achieved in microelectrochemical assays usingPAPP volumes ranging from 20 .mu.l to 360 μL have been reportedpreviously. In capillary assays with electrochemical detection, thelowest detection limit reported thus far is 3000 molecules of mouse IgGusing a volume of 70 μL and a 30 min or 25 min assay time.

Various electrochemical detection systems are described in U.S. Pat. No.7,045,364 (issued May 16, 2006; incorporated herein by reference), U.S.Pat. No. 7,045,310 (issued May 16, 2006; incorporated herein byreference), U.S. Pat. No. 6,887,714 (issued May 3, 2005; incorporatedherein by reference), U.S. Pat. No. 6,682,648 (issued Jan. 27, 2004;incorporated herein by reference); U.S. Pat. No. 6,670,115 (issued Dec.30, 2003; incorporated herein by reference).

IX. Methods of Isolating Virus from a Sample

The invention also encompasses the use of polysaccharide compositionsaccording to the invention for isolating virus from a sample. As usedherein, the term “isolating” refers to separating a desired component(in this case, one or more viruses) from one or more other componentsthat are naturally present with the virus in the sample. This methodexploits the capacity of the polysaccharide composition to bind viruses.Accordingly, the method entails contacting the sample with thepolysaccharide composition of the invention, wherein the polysaccharidecomposition is affixed to a substrate, under conditions suitable for thepolysaccharide composition to bind any virus present in the sample. Thesubstrate is then washed to remove unbound sample material.

The sample can be any type of sample, including, but not limited to, abiological sample, such as any of those described herein. The substratecan be any substrate capable of binding the polysaccharide composition,including any of those described herein. In particular embodiments, thesubstrate is a resin, such as those used for immunoaffinitychromatography. The sample can be contacted with the substrate to allowany virus present to bind to the substrate-affixed polysaccharidecomposition. In particular embodiments, the polysaccharide compositionis reversibly affixed to the substrate. Unbound material can be elutedfrom the column by washing with water or another wash solution which isdesigned to remove at least one component that naturally present in thesample. Bound virus can then be recovered, for example, by releasing thevirus bound-polysaccharide composition from the substrate. Any suitablemeans for reversibly attaching polysaccharides to a substrate can beemployed. See, e.g., Lee, J-C. et al. (2006) Angewandte ChemieInternational Ed. 45:2753-57 (describing the use of a photocleavablelinker to reversibly attach carbohydrates to polymer surfaces), which ishereby incorporated by reference in its entirety.

In other embodiments, after viral binding to a solid phase-affixedpolysaccharide composition of the invention. The bound virus(es) can bedetected and quantified as described above.

As methods for isolating a component from a mixture, based thecomponent's binding capacity are well known, the selection of a suitableformat, substrate, and wash buffer for using the polysaccharidecomposition to isolate virus is within the level of skill in the art.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Preparation of Polysaccharide Composition

A polysaccharide composition of the invention was prepared using seedextract (Spectrum Grape Seed Extract 95% Powder G1273 in the 25 kg size)according to the following protocol.

(1) Prepare a 5% solution (25 gm/500 cc) in distilled water by mixingwith a magnetic stirring rod while slowly adding powder so as not toform surface clumps. Prepare 1000 mL at a time on the Nuova Stir Plateby Thermolyne with the speed set at 8.

(2) Vigorously mix/shake the 5% solution.

(3) Filter the solution with coarse filter paper. Use 4 papers/500 mL;pour 25% into each filter paper (Whatman cat No 1213-185 18.5 cm circle)and then pool the filtrates.

(4) For anion exchange, plug the bottom of a column (3.25 inch diameter,24 inches long, number 4 stopcock) with glass wool and Dowex™ M-43 resin(Dow Chemical) saturated in distilled water to fill the column leaving 4inches to the top free. Prepare the column by back-flow washing withdistilled water so the resin is not packed too tightly in any one area).Load the filtrate obtained in step (3) onto the column gently so as notto disturb the resin. Elute with distilled water and collect all of thecolored material.

(5) Take all colored material from the first column and pass it onto thesecond smaller column (1.5 inch diameter, 24 inches long) filled withthe same resin; elute with distilled water. Collect all the coloredmaterial again. This gives a solution that is almost pure high molecularweight polysaccharide composition of the invention.

(6) The polysaccharide composition can be further purified by removingall the low molecular weight material (less than half a million Daltons)by either dialysis or by using an ion trap or any other means.

(7) The polysaccharide composition can be run through a micropore filterto ensure that it is sterile before putting it into glass ampules orlarge vacuum bottles, for storage as the polysaccharide compositiondeteriorates when exposed to air.

Example 2 NMR Spectroscopy of Polysaccharide Composition

Polysaccharide composition prepared as described in Example 1 wassubjected to NMR spectroscopy.

Methods

NMR Spectroscopy

The sample was exchanged into D₂0 by repeated addition and concentrationof D₂0 (99.9% D) on a rotary evaporator (add 30 mL, evaporate to 2 mL,repeat 6 times). 1-D proton (FIG. 3A) and 2-D gradient enhanced COSY,TOCSY and NOESY NMR spectra (FIGS. 3B-3D) were acquired on a Varian(nova-500 MHz spectrometer at 343 K (70° C.) using standard Varian pulsesequences. Proton chemical shifts are given relative to2,2-Dimethyl-2-silapentane-5-sulfonic acid (DSS; 6=0.00 ppm) andmeasured against the HDO signal (6=4.31 ppm).

Isolation of Non-Carbohydrate Polymer by Methanolysis

Freeze-dried sample (6 mg) was suspended in 1 mL 1 M HCl in MeOH and themixture heated to 80° C. for 6 h in a sealed test tube. After cooling toroom temperature, 1 mL H₂O was added and the methanol was removed bypassing a stream of air over the solution. As the methanol evaporated, athick, red precipitate formed, which was collected by centrifugation.The pellet was washed with water 3 times, and then suspended in 0.7 mLH₂O. Upon addition of 5μL M NaOH the solid dissolved in the water andthe resulting solution was freeze-dried.

Results

The proton spectrum (FIG. 3A) displays at least 10 different anomericprotons, a complex ring region between 3.6 and 4.8 ppm, an intensesinglet at 3.8 ppm, likely due to presence of a methyl ester, and abroad peak between 6.2 and 7.5 ppm. A non-carbohydrate polymer is shownby the broad peak around 7 ppm. This polymer is probably the source ofthe color in the sample.

To separate the non-carbohydrate polymer from carbohydrate, we subjectedthe sample to methanolysis, followed by precipitation and washing, andfound by NMR that the non-carbohydrate is stable to the methanolysisconditions. The NMR of this fraction showed that beside the aromaticpeak two other broad peaks are present at 3.5 and 4.5 ppm (FIG. 3E).

Example 3 Glycosyl Composition of Polysaccharide Composition

Glycosyl composition analysis was carried out as previously described inMerkle and Poppe (1994) Methods Enzymol. 230: 1-15; York, et al. (1985)Methods Enzymol. 118:3-40. Briefly, an aliquot was taken from a sampleof polysaccharide composition prepared as described in Example 1 andadded to separate tubes with 20 μg of Inositol as the internal standard.Methyl glycosides were then prepared from the dry sample following themild acid treatment by methanolysis in 1 M HCl in methanol at 80° C. (8hours), followed by re-N-acetylation with pyridine and acetic anhydridein methanol (for detection of amino sugars). The sample was thenper-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80° C.(0.5 hours). GC/MS analysis of the TMS methyl glycosides was performedon an AT 6890N GC interfaced to a 5975B MSD, using a Supelco EC-1 fusedsilica capillary column (30m×0.25 mm ID). The results are shown in TableA2. Total carbohydrate by weight was found to be 8.2 percent.

TABLE A2 Glycosyl Composition of Polysaccharide Composition Glycosylresidue Mass (: g) Mole % Arabinose (Ara) 24.1 63.0 Rhamnose (Rha) 1.84.3 Xylose (Xyl) 0.9 2.3 Galacturonic Acid (GalA) 4.5 9.2 Mannose (Man)1.1 2.4 Galactose (Gal) 6.6 14.4 Glucose (Glc) 2.0 4.4 E = 41.0 E = 100Total % carbohydrate by weight 8.20%

TABLE A3 Glycosyl Composition of the 150,000 MW Spin-Off ComponentGlycosyl residue Mass (: g) Mole % Arabinose (Ara) 20.3 65.2 Rhamnose(Rha) 1.7 5.0 Xylose (Xyl) 0.6 2.1 Galacturonic Acid (GalA) 3.8 9.5Mannose (Man) 0.9 2.3 Galactose (Gal) 4.2 11.3 Glucose (Glc) 1.7 4.6 E =33.3 E = 100 Total % carbohydrate by weight 5.55%

Example 4 Glycosyl Linkage Analysis of Polysaccharide Composition

A solution of polysaccharide composition prepared as described inExample 1 was placed into a membrane dialysis bag (10,000 MW), dialyzedin deionized water for 3 days, followed by lyophilizing. This sample wasthen methylated by a modification of the method of Hakomori, in whichthe composition was depolymerized, reduced, and acetylated, and theresultant partially methylated alditol acetates (PMAAs) analyzed by gaschromatography-mass spectrometry (GC-MS) as described by York et al(1985) Methods Enzymol. 118:3-40. Briefly, an aliquot was taken from thesample after lyophilizing and dissolved in DMSO; 0.4 mL potassiumdimsylate (2.0 M) was added. After 7 hours at room temperature onstirrer, the reaction mixture was cooled to 0° C., excess methyl iodide(0.7 mL) was added, and the tube sealed. Incubation was then continuedfor 24 h at room temperature. Following sample work-up, thepermethylated material was hydrolyzed using 2 M trifluoroacetic acid (3h in sealed tube at 100° C.), reduced with NaBD₄, and acetylated usingacetic anhydride/pyridine. The resulting PMAAs were analyzed on aHewlett Packard 5890 GC interfaced to a 5970 MSD (mass selectivedetector, electron impact ionization mode); separation was performed ona 30 m Supelco 2330 bonded phase fused silica capillary column.

TABLE A4 Glycosyl Linkage Analysis of Polysaccharide CompositionDK121806 Percentage Glycosyl Residue Present terminally linkedarabinofuranosyl residue (t-Araf) 14.9% 2-linked arabinofuranosylresidue (2-Araf) 9.6% 2-linked rhamnopyranosyl residue (2-Rhap) 0.3%3-linked arabinofuranosyl residue (3-Araf) 3.2% terminally linkedgalactopyranosyl residue (t-Gal) 2.1% 5-linked arabinofuranosyl residue(5-Araf) 15.8% 3-linked glucopyranosyl residue (3-Glc)&2,4-linked 0.7%rhamnopyranosyl residue (2,4-Rhap) 2-linked glucopyranosyl residue(2-Glc) 1.2% 4-linked mannopyranosyl residue (4-Man) 1.4% 3,5-linkedarabinofuranosyl residue (3,5-Araf) 6.9% 2,5-linked arabinofuranosylresidue (2,5-Araf) 5.3% 4-linked glucopyranosyl residue (4-Glc) 4.8%2,3,5-linked arabinofuranosyl residue (3,5-Araf)& 2,3,4- 27.0% linkedarabinopyranosyl residue (2,3,4-Arap) 4-linked galactouronic acid (4-galA) 1.5% 3,6-linked galactopyranosyl residue (3,6-Gal) 0.4%2,3,4,6-linked mannopyranosyl residue (2,3,4,6-Man) 0.7% 2,3,4,6-linkedgalactopyranosyl residue (2,3,4,6- 2.1% Gal)&2,3,4-linked galactouronicacid 2,3,4,6-linked glucopyranosyl residue (2,3,4,6-Glc) 2.2%

The linkage analysis gives information on the type of glycosidiclinkages present in the polysaccharide. The glycosyl composition hadshown that the sample is mainly Arabinose with small amount of anunidentified residue and trace amounts of Mannose, Galactose andRhamnose. This data suggests that the main component in the sample is anarabinan. The linkage data shows that the arabinan consists of backboneof 5-Arabinose and 3,5-Arabinose with side chains of terminal Arabinoseand possibly 2-Arabinose backbone.

Example 5 Binding of Polysaccharide Composition to Herpes SimplexVirus-1 (HSV-1) and Inhibition of Infectivity

The effect of polysaccharide composition prepared as described inExample 1 on HSV-1 infectivity was tested in Vero cells (African GreenMonkey kidney cells). The cells were grown in 12 or 24-well plastictissue culture plates using standard media and conditions. When infectedwith HSV-1, these cells form characteristic plaques which, after 3 days,can be observed by light microscopy. For this study, a 2 log (i.e.,100-fold) dilution of polysaccharide composition was mixed with HSV-1virus at a 2 log dilution in a screw top tube. A second tube containedvirus only, and both were stored in the refrigerator for 11 days. Analiquot from each tube was taken on days 1-11 and assayed. No plaqueswere observed in any well that received the polysaccharidecomposition-virus mixture, whereas plaques were observed in wells thatreceived virus only up until day 11 of storage. These results indicatethat the polysaccharide composition inhibits HSV-1 infectivity for aslong as the virus remains viable.

Example 6 Inhibition of Infectivity of a Broad Range of Viruses byPolysaccharide Composition

The effect of polysaccharide composition prepared as described inExample 1 on HSV-1 infectivity was tested in Vero cells (African GreenMonkey kidney cells). The cells were grown in 12 or 24-well plastictissue culture plates using media and conditions that were standard foreach virus. The results are shown in Tables B1-B4 below and in FIG. 1.

TABLE 1 Effects of undiluted LFT compound on virus infectivity. VirusTiter Virus Titer Log₁₀ with no with Reduction in Virus Family VirusStrain Treatment Treatment Virus Titer Non-enveloped virusesAdenoviridae Adenovirus 1 Chicago 3.5 0^(a) 3.5 Picornaviridae Poliovirus 1 Chat 4.5 0^(a) 4.5 Picornaviridae Rhinovirus 2 HGP 5.5 0^(a) 5.5Reoviridae Human rotavirus Wa 2.25 0^(a) 2.25 Enveloped virusesArenaviridae Tacaribe TV 4.5 0^(a) 4.5 Bunyaviridae Rift Valley Fever(vaccine) MP-12 6.25 0^(a) 6.25 Coronaviridae SARS-CoV Urbani 4.25 0^(a)4.25 Herpesviridae Herpes Simplex 1 McIntyre 4.75 0^(a) 4.75Orthomyxoviridae Influenza A H3N2 A/California/7/04 3.5 0^(a) 3.5Orthomyxoviridae Influenza A H1N1 A/New Caledonia/20/99 4.25 0^(a) 4.25Orthomyxoviridae Influenza A H5N1 Vietnam/1203/4 X Ann 2.75 0^(a) 2.75Arbor/6/60 (H1N1) Orthomyxoviridae Influenza A H5N1 Vietnam/1203/4 X Ann3.75 0^(a) 3.75 Arbor/6/60 (H1N1)- oseltamivir resistantOrthomyxoviridae Influenza A H5N1 DUCK/MN/1525/81 3.5 0^(a) 3.5Orthomyxoviridae Influenza B B/Shanghai/361/02 2.75 0^(a) 2.75Paramyxoviridae Measles MO-6 5.5 0^(a) 5.5 Paramyxoviridae Humanparainfluenza virus 3 14709 3.75 0^(a) 3.75 Paramyxoviridae HumanRespiratory syncytial virus A1 4.75 0^(a) 4.75 Poxviridae Vaccinia virus4.5 0^(a) 4.5 Rhabdoviridae Vesicular stomatitis virus Indiana 4.750^(a) 4.75 Flaviviridae Dengue Fever virus New Guinea FlaviviridaeYellow Fever virus 17D Flaviviridae West Nile virus New York ^(a)Belowdetectable limits of assay.

TABLE 2 Effects of LFT compound diluted 1/10 on virus infectivity. VirusTiter Virus Titer Log₁₀ with no with Reduction in Virus Family VirusStrain Treatment Treatment Virus Titer Non-enveloped virusesAdenoviridae Adenovirus 1 Chicago 3.5 0^(a) 3.5 Picornaviridae Poliovirus 1 Chat 4.5  1.5 3.0 Picornaviridae Rhinovirus 2 HGP 5.5  4.5 1.0Reoviridae Human rotavirus Wa 2.25  1.5 0.75 Enveloped virusesArenaviridae Tacaribe TV 4.5  4.75 0.0 Bunyaviridae Rift Valley Fever(vaccine) MP-12 6.25  6.25 0.0 Coronaviridae SARS-CoV Urbani 4.25  2.252.0 Herpesviridae Herpes Simplex 1 McIntyre 4.75  3.75 1.0Orthomyxoviridae Influenza A H3N2 A/California/7/04 3.5 0^(a) 3.5Orthomyxoviridae Influenza A H1N1 A/New Caledonia/20/99 4.25 0^(a) 4.25Orthomyxoviridae Influenza A H5N1 Vietnam/1203/4 X Ann 2.75 0^(a) 2.75Arbor/6/60 (H1N1) Orthomyxoviridae Influenza A H5N1 Vietnam/1203/4 X Ann3.75 0^(a) 3.75 Arbor/6/60 (H1N1)- oseltamivir resistantOrthomyxoviridae Influenza A H5N1 DUCK/MN/1525/81 3.5 0^(a) 3.5Orthomyxoviridae Influenza B B/Shanghai/361/02 2.75 0^(a) 2.75Paramyxoviridae Measles MO-6 5.5  3.5 2.0 Paramyxoviridae Humanparainfluenza virus 3 14709 3.75  1.25 2.5 Paramyxoviridae HumanRespiratory syncytial virus A1 4.75  3.75 1.0 Poxviridae Vaccinia virus4.5 0^(a) 4.5 Rhabdoviridae Vesicular stomatitis virus Indiana 4.750^(a) 4.75 Flaviviridae Dengue Fever virus New Guinea FlaviviridaeYellow Fever virus 17D Flaviviridae West Nile virus New York ^(a)Belowdetectable limits of assay.

TABLE 3 Effects of LFT compound diluted 1/100 on virus infectivity.Virus Titer Virus Titer Log₁₀ with no with Reduction in Virus FamilyVirus Strain Treatment Treatment Virus Titer Non-enveloped virusesAdenoviridae Adenovirus 1 Chicago 3.5 0^(a ) 3.5 Picornaviridae Poliovirus 1 Chat 4.5 1.5  3.0 Picornaviridae Rhinovirus 2 HGP 5.5 5.75 0.0Reoviridae Human rotavirus Wa 2.25 1.5  0.75 Enveloped virusesArenaviridae Tacaribe TV 4.5 5.25 0.0 Bunyaviridae Rift Valley Fever(vaccine) MP-12 6.25 7.25 0.0 Coronaviridae SARS-CoV Urbani 4.25 3.5 0.75 Herpesviridae Herpes Simplex 1 McIntyre 4.75 4.5  0.25Orthomyxoviridae Influenza A H3N2 A/California/7/04 3.5 1.5  2.0Orthomyxoviridae Influenza A H1N1 A/New Caledonia/20/99 4.25 1.25 3.0Orthomyxoviridae Influenza A H5N1 Vietnam/1203/4 X Ann 2.75 0.75 2.0Arbor/6/60 (H1N1) Orthomyxoviridae Influenza A H5N1 Vietnam/1203/4 X Ann3.75 3.25 0.5 Arbor/6/60 (H1N1)- oseltamivir resistant OrthomyxoviridaeInfluenza A H5N1 DUCK/MN/1525/81 3.5 1.75 1.75 OrthomyxoviridaeInfluenza B B/Shanghai/361/02 2.75 0^(a ) 2.75 Paramyxoviridae MeaslesMO-6 5.5 5.75 0.0 Paramyxoviridae Human parainfluenza virus 3 14709 3.752.75 1.0 Paramyxoviridae Human Respiratory syncytial virus A1 4.75 4.250.5 Poxviridae Vaccinia virus 4.5 2.25 2.25 Rhabdoviridae Vesicularstomatitis virus Indiana 4.75 1.25 3.5 Flaviviridae Dengue Fever virusNew Guinea Flaviviridae Yellow Fever virus 17D Flaviviridae West Nilevirus New York ^(a)Below detectable limits of assay.

TABLE 4 Effects of undiluted LFT compound on virus infectivity whenvirus is diluted by a factor of 10. Virus Titer Virus Titer Log₁₀ withno with Reduction in Virus Family Virus Strain Treatment Treatment VirusTiter Non-enveloped viruses Adenoviridae Adenovirus 1 Chicago 2.5 0^(a)2.5 Picornaviridae Polio virus 1 Chat No infectivity PicornaviridaeRhinovirus 2 HGP 3.75 0^(a) 3.75 Reoviridae Human rotavirus Wa 1.250^(a) 1.25 Enveloped viruses Arenaviridae Tacaribe TV 3.5 0^(a) 3.5Bunyaviridae Rift Valley Fever (vaccine) MP-12 5.25 0^(a) 5.25Coronaviridae SARS-CoV Urbani 3.25 0^(a) 3.25 Herpesviridae HerpesSimplex 1 McIntyre 3.5 0^(a) 3.5 Orthomyxoviridae Influenza A H3N2A/California/7/04 2.5 0^(a) 2.5 Orthomyxoviridae Influenza A H1N1 A/NewCaledonia/20/99 3.25 0^(a) 3.25 Orthomyxoviridae Influenza A H5N1Vietnam/1203/4 X Ann 1.75 0^(a) 1.75 Arbor/6/60 (H1N1) OrthomyxoviridaeInfluenza A H5N1 Vietnam/1203/4 X Ann 1.75 0^(a) 1.75 Arbor/6/60 (H1N1)-oseltamivir resistant Orthomyxoviridae Influenza A H5N1 DUCK/MN/1525/812.5 0^(a) 2.5 Orthomyxoviridae Influenza B B/Shanghai/361/02 1.75 0^(a)1.75 Paramyxoviridae Measles MO-6 4.25 0^(a) 4.25 Paramyxoviridae Humanparainfluenza virus 3 14709 2.75 0^(a) 2.75 Paramyxoviridae HumanRespiratory syncytial virus A1 3.75 0^(a) 3.75 Poxviridae Vaccinia virus3.5 0^(a) 3.5 Rhabdoviridae Vesicular stomatitis virus Indiana 3.750^(a) 3.75 Flaviviridae Dengue Fever virus New Guinea FlaviviridaeYellow Fever virus 17D Flaviviridae West Nile virus New York ^(a)Belowdetectable limits of assay.

The polysaccharide composition appears not to act via binding to areceptor on cells because when the polysaccharide composition is addedto cells, followed by washing and subsequent exposure to virus, therewas no observed inhibition of infectivity. In addition, exposure of thepolysaccharide composition to cells for 10 minutes did not reduce itsability to inhibit viral infectivity. These findings indicate that theeffect of the polysaccharide composition on viral infectivity is due toits binding virus, as opposed to binding cells or some other componentor uptake by cells.

The antiviral activity of polysaccharide composition was assessed in acytopathic effect and plaque reduction assay reduction (CPE) assay, andthe composition was found to have specific antiviral activity againstboth HSV-1 and HSV-2. To confirm these data, a standard plaque reductionassay was used to measure antiviral activity and no activity wasobserved. These results suggested that the extract might need to bepresent during the attachment phase of viral replication. When the studywas repeated and the cells were pretreated with the polysaccharidecomposition, antiviral activity was observed although it was lesseffective than the activity observed in the CPE assay. This confirmedthat the polysaccharide composition was acting during the attachmentphase of viral replication. It was also possible that the extract mightbe directly inactivating virus particles prior to attachment, so astandard inactivation study was used to evaluate this potentialactivity. This study also confirmed that the polysaccharide compositioncan directly inactivate virus particles. These data taken togethersuggest that the polysaccharide composition acts prior to infection andcan inactivate virions as well as block infection during attachment.

Example 7 In Vivo Protection Against Viral Challenge by PolysaccharideComposition-Based Vaccine

In this study, the polysaccharide composition of the invention, preparedas described in Example 1 (referred to herein as “LFT compound”) wastested for ability to confer in vivo protection against viral challenge.

Introduction

Example 6 shows that LFT compound can inactivate a wide range ofenveloped and non-enveloped viruses, including avian influenza Aviruses. It has been hypothesized that the interaction of LFT compoundwith each virion, which leads to the inactivation of infectious virionscould be useful in developing an inactivated vaccine, especially if theinteraction between the LFT compound and the virion did not alter thekey immune recognition sites on the virion necessary for eliciting of aprotective response to infectious virus. The purpose of the currentresearch reported below was to test the hypothesis that avian influenzaA H5N1 virus inactivated by LFT compound could be used to protect miceagainst lethal infection by influenza A/Duck/MN/1525/81 (H5N1) virus.

Materials and Methods

Virus and Cells

Madin Darby canine kidney cells (MDCK) were obtained from the AmericanType Culture Collection (ATCC, Manassas, Va., USA). Growth medium wasminimal essential medium (MEM) with 5% fetal bovine serum (HycloneLaboratories. Logan, Utah, USA) supplemented with 0.1% NaHCO₃. Growthmedium had no supplemental antibiotics. When evaluating virusreplication in the cells, the medium was MEM without serum, 0.18%NaHCO3, 20 μg trypsin/ml, 2.0 μg EDTA/ml, and 50 μg gentamicin/ml.Influenza A/Duck/MN/1525/81 (H5N1) was obtained initially from Dr.Robert Webster of St. Jude Hospital, Memphis, Tenn. It was eitherpassaged in MDCK cells in vitro, or it was passaged through mice untiladapted to the point of being capable of inducing pneumonia-associateddeath in the animals. Pools of the virus were subsequently prepared foranimal studies or cell culture studies and were maintained at −80° C.

Inactivated Virus for Vaccine Study

Influenza A H5N1 virus (MN/1281/81) at 10^(5.5) TCID₅₀, 10^(4.5) TCID₅₀,or 10^(3.5) TCID₅₀ was treated with LFT lot #2 for 24 h at 37° C. Afterthe treatment, the preparations were aliquoted and stored at −80° C.until used to immunize animals.

Reagents

LFT (lot #2) was used for this study. Alum was obtained from PierceBiotechnology, Inc. (Rockford, Ill., USA).

Animals

Female specific pathogen-free 18-21 g BALB/c mice were obtained fromCharles River Laboratories (Wilmington, Mass.). They were quarantined 5days prior to use. They were housed in polycarbonate cages withstainless steel tops and provided tap water and standard rodent mousechow ad libitum.

Vaccine Challenge Study

Groups of 10 mice were immunized with one of three concentrations(10^(5.5) TCID₅₀, 10^(4.5) TCID₅₀, or 10^(3.5) TCID₅₀) of LFT-treatedvirus intramuscularly (i.m.) or intradermally (i.d.) or with PBS i.m. ori.d. in the presence or absence of alum. Immunizations were given ondays 0, 14, and 28. On days −7, 21 and 42 and 56 relative to exposure toinactivated virus vaccine, serum was collected by submandibular bleedfrom all living mice for measurement of neutralizing antibody. On day42, mice were challenged intranasally with 10³⁵ TCID₅₀ of influenza AH5N1 virus (MN/1281/81). On day 46, 3 mice from each group weresacrificed to harvest lungs for assessment of virus lung titers; theremaining, surviving mice were sacrificed on day 56 to harvest lungs forvirus titration and blood for virus neutralization tests.

Neutralizing Antibody Assay

An equal volume of a serum sample at an appropriate dilution (usually1:50 or 1:100) and virus at 200 TCID₅₀ were mixed and incubated at 37°C. for 1 hour under gentle rocking conditions. At the end of this time,the neutralization mixture was serially diluted and the surviving virustitered by CPE (cytopathic effect) assay. Eight dilutions were plated inquadruplicate and the assay was done three times on the same plate foreach serum. For the CPE assay, 0.1 ml of a neutralization sample wasadded directly to the cell culture plate containing cells at theappropriate cell density (1×10⁴ Vero 76 cells/well) plated the previousday in 96-well plate. An additional 0.1 ml of medium, containing 20 μgtrypsin/ml, 2.0 μg EDTA/ml, and 50 μg gentamicin/ml (all finalconcentrations) was then added to each well, gently mixed and incubatedat 37° C. for 6 days, the optimal time required to achieve fullcytopathic effect in the non-treated infectivity controls when using thevirus at 200 TCID₅₀ units. The wells in the plate were then scored byvisual lobservation for cytopathic effect or cytotoxicity using lightmicroscopy. CPE was graded upon a scale of 0-4; 0=no cytopathic effectand 4=100% cytopathic effect. Titers were then calculated using theReed-Muench method. The inverse of the most dilute serum samplecompletely protecting cells from virus cytopathic effects was consideredthe virus neutralization titer for the serum. Infectivity controls virustitrations (“virus back titration”), which were an equal volume of virusadded to an equal volume of MEM and treated in the same manner as thesera were titered last as a control for virus deterioration during theassays of the sera.

Lung Scoring

Lungs were scored based on surface appearance of lungs. Lungs wereassigned a score from 0-4, with 0 meaning that the lungs looked normaland 4 denoting that the entire surface area of the lung was inflamed andshowed plum colored lung consolidation.

Lung Virus Titer Determination

Each mouse lung was homogenized, the tissue fragments allowed to settle,and varying dilutions of the supernatant fluids were assayed intriplicate for infectious virus in Vero 76 cells by CPE assay and titers(TCID₅₀ values) calculated using the Reed-Muench method.

Histopathology

Thin sections of lungs were stained with hematoxylin and aboard-certified veterinarian for pathology examined eosin and theslides.

Statistical Analysis

Differences in mean lung virus titers were evaluated by the analysis ofvariance. Differences in death rates were determined by Chi-squareanalysis.

Results

The results are shown in Table C1 below.

TABLE 1 Effects of Immunizing Mice with LFT-Inactivated InfluenzaA/Duck/MN/1525/81 (H5N1) Neutralizing Antibody Titer* (Inverse ofDilution ± SD) Live/ Vaccine Day of Bleed Relative to Vaccine LUNG VIRUSTITER, log₁₀ Total (Amount of Administration CCID50 ± SD Day TypicalHistopathogical GROUP inactivated virus) 0 21 46 56 46 56 56 Descriptionfor Group Vaccine without Adjuvant (3 animals, day 46) Intramuscularadministration (i.m.) 1 (10^(5.5)CCID₅₀) <50† <50 <50 700 ± 589 5.96 ±0.07   0.53 ± 0.06 4/7^(¶) Severe inflammation 2 (10^(4.5)CCID₅₀) <50<50 <50 433 ± 197 6.00 ± 0.13   0.54 ± 0.07 3/7 Moderatebronchopneumonia 3 (10^(3.5)CCID₅₀) <50 <50 <50 400 ± 0  5.83 ± 0.19<0.50 ± 0.00 1/7 Mild-Mod bronchopneumonia 4 PBS <50 <50 <50 Nosurvivors 5.75 ± 0.43 No survivors 0/7 Mild-Mod bronchopneumoniaIntradermal administration (i.d.) 5 (10^(5.5)CCID₅₀) <50 <50 <50 667 ±197 5.67 ± 0.29 <0.50 ± 0.00 6/7^(‡) Severe inflammation 6(10^(4.5)CCID₅₀) <50 <50 <50 667 ± 197 5.79 ± 0.64 <0.50 ± 0.00 6/7^(‡)Mild bronchopneumonia 7 (10^(3.5)CCID₅₀) <50 <50 <50 450 ± 252 6.04 ±0.52 <0.50 ± 0.00 2/7 Mild-Mod bronchopneumonia 8 PBS <50 <50 <50 400 ±0  5.79 ± 0.19 <0.50 ± 0.00 3/7 Mild bronchopneumonia Vaccine + Alumadjuvant Intramuscular administration 9 (10^(5.5)CCID₅₀) <50 <50 75 ± 431235 ± 1570 4.71 ± 0.69^(‡) <0.50 6/6^(‡) Moderate bronchopneumonia 10(10^(4.5)CCID₅₀) <50 <50 108 ± 101 1494 ± 1885 5.63 ± 0.00 <0.50 ± 0.004/7^(¶) Mid-Mod bronchopneumonia 11 (10^(3.5)CCID₅₀) <50 <50 <50 Alldead 6.17 ± 0.59 <0.50 ± 0.00 0/7 Mild-Mod bronchopneumonia 12 PBS +Alum (i.m.) <50 <50 <50 267 ± 230 6.21 ± 0.29 <0.50 1/7 Moderatebronchopneumonia Intradermal administration 13 (10^(5.5)CCID₅₀) <50 <50450 ± 260 242 ± 186 1.88 ± 1.85^(¶) <0.50 ± 0.00 7/7^(‡) Moderatebronchopneumonia 14 (10^(4.5)CCID₅₀) <50 <50 <50 535 ± 2 5.63 ± 0.33<0.50 ± 0.00 3/7 Mild-Mod bronchopneumonia 15 (10^(3.5)CCID₅₀) <50 <50<50 533 ± 462 6.13 ± 0.38 <0.50 ± 0.00 1/7 Mild inflammation 16 PBS +Alum <50 <50 <50 334 ± 94 5.54 ± 0.38 <0.50 ± 0.00 2/7 Mild-Modbronchopneumonia Toxicity Controls-No Virus challenge 17 PBS + Alum(i.m.) <50 <50 <50 <50 <0.50 <0.50 ± 0.00 2/2 No lesions 18 Vaccine +Alum (i.d.) <50 <50 400 ± 0  267 ± 189 <0.50 <0.50 ± 0.00 2/2 No lesions19 Vaccine + Alum (i.m.) <50 <50 <50 <50 <0.50 <0.50 ± 0.00 2/2 Nolesions *Neutralizing titer = largest antibody dilution that preventedvirus cytopathic effect. †Denotes that virus neutralization titer wasbelow the limit of detection of assay. ^(‡)Significantly different fromplacebo control, P < 0.01. ^(¶)Significantly different from placebocontrol, P < 0.001.

The alum and the vaccine appeared to be well-tolerated when evaluated inmice without wild type virus challenge; they did not induce detectablepathogenesis in the lungs (See groups 17-19, Table C1).

Without Adjuvant

Intramuscular

Intramuscular administration of the whole unit inactivated virus vaccinewithout adjuvant was effective in protection against challenge from wildtype virus. Four of seven animals survived, although the virus lungtiters in these animals were not reduced relative to the placebo control(Table C1). This may have been the reason that the lungs of survivinganimals seemed to be characterized by severe inflammation. Modestamounts of neutralizing antibody (titer=700) were generated at thehighest dose of vaccine used. Administration by intradermal route usingthe vaccine without adjuvant was also protective at two highest dosagesused with six of seven animals surviving. However, virus lung titerswere not reduced compared to unimmunized animals and the lungs ofanimals receiving the largest dose of vaccine were again characterizedby severe inflammation. However, the lungs of animals receiving ten-foldless inactivated virus particles were characterized by much less severepathogenesis. Modest amounts of neutralizing antibody (titer=667) weregenerated at the two highest doses of vaccine used. Some neutralizingantibody was also generated by exposure to the infectious virus itself.

With Adjuvant

Administration of vaccine in the presence of alum adjuvant appeared tobe the most effective in terms of not only in protecting against deathto due to virus challenge, but also in ameliorating lung pathogenesisand lowering virus lung titers.

Intramuscular and Intradermal

Animals treated with highest dose of vaccine, administered either i.m.or i.d., were all protected against death (i.m., 6/6; i.d., 7/7), thevirus titers were significantly reduced (i.m., P<0.01; i.d., P<0.001)and the lung pathogenesis was not as severe as in animals immunized withvaccine only and protected against death. In addition, the i.d.immunization appeared to be the most effective of all immunizations inreducing virus lung titers (3.66 log₁₀ reduction).

Discussion

The data indicate that any animal that survives the lethal infectionwith challenge virus will produce neutralizing antibody (Table C1). Inthe presence of high doses of the inactivated vaccine, production ofneutralizing antibody seemed to increase as did survival to lethalchallenge of virus, a phenomenon which has been shown with othervaccines against influenza, for example with a vaccine against the 1918strain of influenza A. The sole exception to this generalization was thevaccine regimen in which high dose inactivated vaccine in adjuvant,administered i.d was extremely protective, but did not result in muchneutralizing antibody being produced. However, virus lung titers inanimals treated with this vaccine were greatly reduced by almost afactor of 10,000. It may be that this particular vaccine regimen alsostimulated other protective, immune responses such as interferon andnatural killer cell activity very early in the challenge infection thatresulted in the substantial elimination of infectious virus particles tobe presented to antigen presenting cells. This early elimination ofvirus infectious virus may have significantly reduced the amount ofvirus available for antigen presentation and thus reduced the number ofB cells programmed to produce neutralizing antibody. Intradermaladministration of the whole unit inactivated virus vaccine with orwithout adjuvant seemed to be somewhat more protective against deaththan i.m. administration. This could be due to the fact underneath theskin are dendritic cells (Langerhans cells), one of the major antigenpresenting cells of the body, while muscle is not considered to be asite for antigen presentation because it contains few if any dendriticcells. It may be that in this experiment, better antigen presentationoccurred due to direct interaction of dendritic cells underneath theskin with the antigen present in the vaccine. This in turn could haveled to an increased, proper presentation of the appropriate antigen tothe immune system resulting in a more efficient and quicker response toinfection upon challenge with live virus.

Administration of vaccine in the presence of alum adjuvant appeared tobe the most effective regimen in terms of not only protecting againstdeath due to virus challenge, but also in ameliorating lung pathogenesisand lowering virus lung titers. Adjuvants such as alum cause whatamounts to be a time-release of antigen to the immune system, leading toenhanced uptake by phagocytic antigen presenting cells such asmacrophages and dendritic cells. The results of such a release andenhancement could have also led to the enhanced immune response seen inthe current study.

Conclusions

The intramuscular administration of vaccine in alum adjuvant was overallthe most effective vaccine regimen used. It protected 100% of the micefrom death due to virus challenge, limited lung pathogenesis,significantly reduced the virus lung titers in treated animals andstimulated the production of the greatest amount of neutralizingantibody. Intradermal immunization in the presence of adjuvant might beconsidered even more efficacious, if neutralizing antibody weregenerated in greater amounts. These results suggest that LFT-inactivatedvirus represents a new method of creating inactivated vaccines thatshould be pursued.

Example 8 Evaluating Adjuvant-Free Polysaccharide CompositionInactivated Influenza A/Duck/MN/1525/81 (H5N1) Virus Vaccines in aLethal BALB/c H5N1 Influenza A Model

Purpose of the Study

The purpose of the study was to test the hypothesis that avian influenzaA H5N1 virus that had been inactivated by a polysaccharide compositionof the invention (prepared as described in Example 1), administeredintradermally or intranasally, could be used to protect mice againstlethal infection by influenza A/Duck/MN/1525/81 (H5N1) virus withoutusing an adjuvant. This study employed the same general methods andtypes of animals as Example 7.

Summary

The polysaccharide composition-inactivated (10 min) influenzaA/Duck/MN/1525/81 (H5N1) vaccine was completely protective againsthomologous virus challenge at certain doses without the use of adjuvant.Intranasally administered vaccine, given twice 14 days apart, was themost efficacious immunization regimen. The data suggest thatpolysaccharide composition represents an innovative and effective way ofderiving vaccines.

Vaccine Regimen

“Vaccine 1a”=With 10 min. inactivated virus at 10^(5.7) CCID₅₀administered twice intranasally.

“Vaccine 1b”=With 10 min. inactivated virus at 10^(4.7) CCID₅₀administered twice intranasally.

“Vaccine 1c”=With 10 min. inactivated virus at 10^(3.7) CCID₅₀administered twice intranasally.

Each of the above vaccines was prepared as described in Example 7,except that the polysaccharide composition was exposed to virus for 10minutes, rather than 24 hours.

Survival

Two courses of Vaccine 1a and 1b (polysaccharide composition-inactivatedfor 10 min) delivered intranasally significantly protected micecompletely, although Vaccine 1a prevented death in all immunized mice.

Neutralizing Antibody

In mice receiving Vaccines 1a, 1b, and 1c twice intranasally, there weresubstantial amounts of neutralizing antibodies two days prior to viruschallenge and none in mice receiving no vaccine as had been expected.Three days after challenge with virus, the neutralizing antibody beganto rise in the mice receiving Vaccines 1a and 1b, and, in general, thesetiters continued to rise to the end of the trial.

Virus Lung Titers

Interestingly, mice immunized twice intranasally with Vaccine 1a had nodetectable virus lung titers, which corresponded with a 100% survivalrate in this group and the highest virus neutralization titers.

Effects of Various Lung Parameters

It was assumed that if the vaccine prevented or lessened viralcolonization of lung tissue, it should result in reduced lung scores,lung pathology, lung weights, and better lung function as manifested inhigher saturated blood oxygen levels.

Lung Scores

Lung scores for mice immunized twice with Vaccine 1a intranasally weresignificantly lower on days 6 and 14 than those lung scores recorded forunimmunized mice on the same days after virus challenge (P<0.05-0.01).This corresponded with significant protection against death.

Lung Pathology

The lung pathology of mice immunized twice intranasally was notremarkably different than the lung pathology observed for theunimmunized cohort mice at day 3. In general, for all groups, thepathology was characterized by a few bronchioles segmentally lined bynecrotic epithelial cells containing luminal neutrophils. A few largeairways were surrounded by small aggregates of lymphocytes, some formingfollicles.

Arterial Saturated Oxygen (SaO₂) Levels

In the current study, all of the unimmunized mice for each vaccine trialarm had much lower SaO₂ level trends than did the immunized cohorts,regardless of vaccine used or the route of delivery. When Vaccine 1a wasgiven twice intradermally or intranasally, SaO₂ levels in these miceremained significantly higher when compared to the SaO₂ levels in therespective cohorts for each trial arm. This correlated well withsurvival rates, especially for mice receiving Vaccine 1a intranasally.

Weight Changes

One parameter that is often associated with severe disease and sometimeswith death in the influenza mouse model used in the current vaccinestudy is weight loss. Mice receiving Vaccine 1 once or twiceintranasally, which were some of the more effective vaccine regimensprotecting mice against lethal virus challenge, also had the leastamount of weight change throughout the duration of the experiment. Theyalso had less weight loss.

Cytokine Levels

For mice receiving Vaccine 1 twice intranasally at day 6, only thelevels of the chemokine MCP-1 and the cytokine TNF-alpha seemed to bedramatically reduced compared to levels of that chemokine and cytokinein unimmunized mice.

All levels for all cytokines and chemokines were nearly the same inimmunized and in the unimmunized animals with the exception of IL-12levels in unimmunized mice, which were much higher. The apparentreduction of IL-12 levels in immunized mice correlated with thesignificant increase in immunized mice surviving virus infection.

The cytokine “storm” one often expects with an H5N1 infection with micewas not detected in this vaccine study with the exception of in theunimmunized mice, which had drastically higher IL-12 levels.Immunization apparently reversed IL-12 levels in mice receiving thevaccine.

Discussion

The polysaccharide composition vaccines were well tolerated throughoutthe immunization period, regardless of the number of doses given,indicating a lack of overt toxicity.

For efficacy of protection, the best vaccine and regimen evaluated wasusing the vaccine containing 10^(5.7) virus inactivated bypolysaccharide composition for 10 min, delivered intranasally twice withthe second dose coming 14 days after the primary vaccine dose. The virusneutralization titers of mice immunized with the twice-deliveredintranasal vaccine were highest 14 days after virus challenge.

The vaccine eliminated the colonization of the lung by challenge virus.This translated into less gross pathology and milder pathology detectedin sectioned lungs.

The levels of proinflammatory cytokines detected in the lungs of theseanimals were also substantially lower than in other immunized group ofmice, reinforcing the conclusion that the vaccine containing 10^(5.7)virus inactivated by polysaccharide composition for 10 min, deliveredintranasally twice prevented severe lung pathology due to virusinfection. These effects probably are indicative of near normal lungfunction as well. This conclusion is supported by the fact the arterialsaturated oxygen levels in this immunized group of mice weresubstantially higher than those detected in the unimmunized mice andmost other immunized groups of mice.

Inactivation of the virus with polysaccharide composition for only 10minutes was sufficient to reduce the levels on infectious virus to theundetectable limits of the assay used to quantitate infectious virus(data not shown), yet was sufficient to elicit high titers ofneutralizing antibody.

Conclusions

The results suggest that an effective vaccine can be made frompolysaccharide composition-inactivated virus after only a 10 minuteexposure, that it can be delivered without adjuvant, unlike previouslyconjectured, and that intranasal delivery provides the most protectiveresponse against death, pathology, localized detrimental cytokineincreases, and virus colonization by challenge virus.

The efficacy seen with the current polysaccharidecomposition-inactivated vaccine given intranasally and without adjuvantwas achieved with two exposures and no adjuvant. In addition to reducedlung pathogenesis or no lung pathogenesis and little or no viruscolonization of the lung by challenge virus, neutralizing antibodytiters were high. This suggests that polysaccharide compositioninactivated vaccines without adjuvant are efficacious without theproblems associated with adjuvant formulation.

Thus, polysaccharide composition-inactivated virus represents a newmethod of creating inactivated vaccines that should be pursued forpossible use in developing a new human influenza A vaccine. Thetechnology is be amenable for creating inactivated vaccines for otherviral diseases.

Example 9 The Polysaccharide Composition Inhibits Growth of Cancer Cells

The treatment of cancer cell lines with polysaccharide compositionprepared as described in Example 1 inhibits replication of variouscancer cells. In this study, Vero cells, A549 (human lung carcinoma),HeLa229 (human cervix adenocarcinoma), NCI-H292 (pulmonarymuco-epidermoid carcinoma), and (CACO-2: human colon carcinoma) wereexposed to various concentrations of the polysaccharide composition togenerate dose-response curves indicating that the polysaccharidecomposition can inhibit and even prevent replication and that thiseffect correlated with dose. The effect was reversible if the cells werewashed within 3 hours of initial exposure. After 3 hours of exposure,the cells would not replicate and went on to die. If the polysaccharidecomposition was washed off within 3 hours, cell division restarted in4-6 hours.

Example 10 Treatment of Squamous Cell Carcinoma with a PolysaccharideComposition

Topical Administration

A human subject having a squamous cell carcinoma on the chin was treatedwith a polysaccharide composition prepared as described in Example 1.FIG. 4A shows the lesion as it appeared before treatment. The treatmentregimen consisted of topical application of the polysaccharidecomposition every 15 minutes for a period of 4 hours; this treatment wascarried out once daily. The appearance of the lesion on treatment day 5is shown in FIG. 4B. Whereas the lesion included a large, raised moundprior to treatment, at day 5, the lesion included a much smaller raisedarea, and the edges of the lesion are beginning to heal inward. Inaddition, comparison with the blue dot (¼ inch in diameter) indicatesthat the lesion is smaller. FIG. 4C shows the lesion on treatment day 9.At day 9, the lesion appeared smaller and flatter than on day 5,indicating that the healing process was continuing.

Oral Administration

Oral administration of the polysaccharide composition, prepared asdescribed in Example 1, was also studied. An 89-year-old male subjectreceived 150 ml daily of approximately 40 mg/ml polysaccharidecomposition, taken in divided doses over 4 hours. After 20 oraltreatments, the skin cancer was significantly reduced (see FIG. 5).

A 94-year-old female subject with terminal metastatic squamous cellcarcinoma was given one treatment of the polysaccharide compositionorally (about 100 ml of approximately 40 mg/ml taken in divided dosesover 4 hours). Measurements were preformed on the left posterior neckregion. The measurable external part of the tumor was initially 1.75inches in diameter on the evening of 7-26-08, then 1.5 inches indiameter on the morning of 7-27-08, and 1.25 inches on the evening of7-28-08. On 8-1-08, the tumor was ⅞ inches in diameter with necroticareas. See FIGS. 6A-C.

Example 12 Treatment of Chronic Lymphocytic Leukemia with aPolysaccharide Composition

An 87-year-old male subject with chronic lympocytic leukemia was treatedwith the polysaccharide composition prepared as described in Example 1.Specifically, each day, 4 drops were applied to surface skin every 15minutes for a duration of 4 hours.

The initial blood count and cytology are shown below.

Collected Date: Aug. 21, 2006 15:10:00 Case #: SP-06-0007883

Diagnosis

Peripheral Blood for Flow Cytometric Analysis:

-   -   B-cell chronic lymphocytic leukemia.    -   Immunophenotype: CD5, CD19, CD20, CD22, CD23, kappa positive.

-   22 Aug. 2006 Pam Suwanjindar, MD

-   LSR/PS (Electronic Signature)    Comments

-   In addition to CLL, the peripheral smear and CBC data show findings    suggestive of iron deficiency anemia.    Specimen

-   Peripheral blood for flow cytometry    Clinical Information

-   The patient is an 86-year-old male with absolute lymphocytosis.

-   CBC DATA: WBC 20.6 with 72% lymphocytes, hemoglobin 8, hematocrit    30, MCV 38, MCHC 27, and platelets 221.    Gross Description

-   Received from Dr. Kenyon through Legacy Bridgeview Laboratory,    Newport, Oreg. is an EDTA tube of peripheral blood for flow    cytometry studies. A Wright-stained peripheral smear is prepared and    reviewed for morphologic correlation with flow cytometry results.

-   PS/lr    Microscopic

-   A Wright-stained peripheral smear demonstrates anisopoikilocytosis    with scattered ovalocytes, microcytes, schistocytes, targets cells,    and hypochromic cells. The WBC count is increased with an absolute    lymphocytosis. The majority of lymphocytes are small with a    condensed chromatin pattern. No significant prolymphocytes are seen.    Platelets are adequate.

-   P S/lr

The post-treatment blood count and cytology follow. These show loss ofCD20 marker with treatment, as well as reduction of white blood cells(WBC).

Surgical Pathology

-   Collected Date: Jul. 17, 2008 16:20:00 Case#: SP-08-0007308    Diagnosis    Peripheral Blood for Flow Cytometric Analysis:    -   B-cell chronic lymphocytic leukemia.    -   CD5, CD19, CD23, kappa positive and CD38 negative (see        Comments).-   18 Jul. 2008 Pam Suwanjindar, MD-   MJC/PS (Electronic Signature)    Comments-   The neoplastic B-cells do not express CD20 which may be due to    previous chemotherapy.    Specimen-   Peripheral blood for flow cytometry    Clinical Information-   88-year-old male with history of B-cell chronic lymphocytic leukemia    (CD5, CD19, CD20, CD23 and kappa positive).-   CBC: WBC 9.3 with absolute lymphocyte count 5.0, hemoglobin 9,    hematocrit 31, MCV 102, RDW 15, platelets 206.    Gross Description-   Received from Samaritan Hematology and Oncology Consultant,    Corvallis, Oreg., is an EDTA tube of peripheral blood for flow    cytometry. A Wright—stained peripheral smear is prepared and    reviewed for morphologic correlation with the flow cytometry    results.    Microscopic-   A Wright-stained peripheral smear demonstrates no significant    anisopoikilocytosis. The WBC count is within the normal range with    an absolute lymphocytosis of 5.0. The majority of lymphocytes are    small with a condensed chromatin pattern. No significant large    lyphoid cells or prolymphocytes are seen. Platelets are adequate.-   PS/mjc

Example 13 Effects Treatment with a Polysaccharide Composition onVarious Parameters Associated with Aging

Hair Restoration

Oral Administration

An 89-year-old male subject with apical balding for 30 years, where theremaining hair was white in color, received a 3-week oral treatment of150 ml daily of approximate concentration of about 40 mg/mlpolysaccharide composition (prepared as described in Example 1), takenin divided doses over 4 hours. After 3 weeks, this subject regrew hairin the bald area and developed 50% black hair, where he originally hadwhite hair only. The color of the regrown hair was black, the color ofthis man's hair when young. See FIG. 7.

Topical Administration

With topical treatment twice daily of a solution 40 mg/ml of thepolysaccharide composition, a 54-year-old male subject with mostly greyhair and a receding hair line re-established hair in the areas ofthinning and recession. New hair was observed in the frontal hair linethat was previously bald. The new hair was brown, rather than grey.Apical bald area was reduced from a diameter of 3 inches with new hairthat brown. Mostly grey hair changed to color to brown over a 1-yeartreatment period.

Sub-Lingual Administration

Sub-lingual daily doses of approximately 20 mg over a 6-month periodconverted the hair color in a 68-year-old male subject from pure whiteto approximately 30% black and 70% white.

Hormonal Changes

Sub-lingual dose of 60 mg over a 4 hour period daily for 14 days changedthe FSH and estradiol in a 63 year old woman as follows:

-   Pre-treatment: FSH: 78.5; Estradiol: 27-   Post-treatment: FSH: 49.6; Estradiol: 23    Fat Loss

Three human subjects taking daily doses of approximately 20 mgpolysaccharide composition noted reduced fat on the abdomen area afterseveral weeks without changes in diet or exercise. This fat losscontinued over a 3-month observational period.

Liver Spots

Topical application of a 40 mg/ml solution of polysaccharide compositionto “liver spots” on the hands of a 55 year old man twice dailydiminished and removed the spots over a few days of application, as wellas improving elasticity and skin thickness.

Near Vision Improvement

Sub-lingual daily doses of approximately 20 mg polysaccharidecomposition produced near vision improvement in a 55-year-old malesubject from over 1000 mm down to 140 mm for closest non-blurred visionover a 6-month treatment.

Improved Stamina, Muscle Mass, and Cognition

Three people (a 55-year-old man, a 68-year-old man, and an 85-year-oldman) all taking sub-lingual doses of approximately 20 mg polysaccharidecomposition reported both subjective and objective improved mentalfunction. Improvements were noted in memory, arithmetic skills, andlogic. Stamina in exercise and muscle mass also improved slowly overseveral weeks.

Example 14 Additional Characterization Studies of PolysaccharideComposition

A polysaccharide composition according to the invention is referred toas “Galahad” in this example. A component of the polysaccharidecomposition is a red material that can be precipitated by addition ofammonium sulfate and can thus be separated from the carbohydrate in thecomposition. This red material is referred to as “Galahad Red” in thisexample.

Methods

Glycosyl Composition

Glycosyl composition analysis was performed by combined gaschromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl(TMS) derivatives of the monosaccharide methyl glycosides produced fromthe sample by acidic methanolysis.

Methyl glycosides were first prepared from dry sample provided by theclient by methanolysis in 1 M HCl in methanol at 80° C. (18-22 hours),followed by re-N-acetylation with pyridine and acetic anhydride inmethanol (for detection of amino sugars). The samples were thenper-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80° C.(0.5 hours). These procedures were carried out as previously described(Methods Enzymol. 230:1-15; York W. S., Darvill, A. G., McNeil, M.,Stevenson, T. T., and Albersheim, P. (1985) Methods Enzymol. 118:3-40).GC/MS analysis of the TMS methyl glycosides was performed on an HP 5890GC interfaced to a 5970 MSD, using a All Tech EC-1 fused silicacapillary column (30m×0.25 mm ID).

Glycosyl Linkage

Hakamori Method:

For glycosyl linkage analysis, the dialyzed (10 kDa) sample wasmethylated by a modification of the method of Hakomori; depolymerized,reduced, and acetylated; and the resultant partially methylated alditolacetates (PMAAs) analyzed by gas chromatography-mass spectrometry(GC-MS) as described by York et al (1985) Methods Enzymol. 118:3-40.Briefly, An aliquot was taken from the sample after lyophilizing anddissolved in DMSO, 0.4 mL potassium dimsylate (2.0 M) was added. After 7hours at room temperature on stirrer, the reaction mixture was cooled to0° C., excess methyl iodide (0.7 mL) was added, and the tube sealed.Incubation was then continued for 24 h at room temperature. Followingsample workup, the permethylated material was hydrolyzed using 2 Mtrifluoroacetic acid (3 h in sealed tube at 100° C.), reduced withNaBD₄, and acetylated using acetic anhydride/pyridine. The resultingPMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970MSD (mass selective detector, electron impact ionization mode);separation was performed on a 30 m Supelco 2330 bonded phase fusedsilica capillary column.

Size Exclusion Chromatography

Prior to loading, hydrophobic compounds were removed by C18chromatography. Sample was loaded onto a Scc Waters C18 Sep Pak in 5%acetonitrile and eluted with water. A significant amount of red colorremained at the top of the column and was eluted with 100% acetonitrile.This amounted to some 3 mg. Load and water wash were pooled andlyophilized to get approximately 20 mg of material. Interestingly, thered colored material which remained in the load was not soluble whenresuspended at 10 mg/ml in 50 mM ammonium formate pH 5.06 and wasretained when the material was passed through a 0.45 micron filter priorto chromatography. Filtered sample was loaded onto a 0.8×30 cm ToyopearlHW65 size exclusion column and eluted at 1.0m|/min in 50 mM ammoniumformate pH 5.06. Detection was accomplished by means of an Agilentrefractive index detector. Retention times were compared to standards of1400 kD, 511 kD, 167 kD and 40 kD dextran.

Dynamic Light Scattering

Dextran standards (40, 167, 511, and 1400 kDa) and ammonium sulfateprecipitated and dialyzed Galahad were prepared as 2 mg/mL solutions inwater. The raw Galahad solution was diluted 5-fold with water. Thesamples were centrifuged for 15 min before analysis to removeparticulates.

Measurements were carried out on a Protein Solutions DynaPro 99 dynamiclight scattering machine at 25° C., using the Dynamics software, version6.03. Laser power was set to 50% (standards) and 30% (samples),respectively. Sampling interval was 5 s and a minimum of 20 measurementswas collected and averaged.

Molecular Weight Determination Using SDOC PAGE of Galahad

Electrophoresis was performed on 8×10 cm 18% T 2.7% C mini gels using aTris/Glycine (1:4.8 w/w) 0.25% sodium deoxycholate running buffer at 30mA current and 400V potential. The gels were fixed in 40% ethanol 5%acetic acid 0.005% alcian blue 60 minutes then reincubated in the samesolution overnight. Gels were then rinsed in water and oxidized in 0.7%sodium metaperiodate for 10 minutes. After this, they were rinsed 5times in water and incubated 10 minutes in 10% Bio Rad silver reagentconcentrate, rerinsed in water and developed in 3.2% Bio Rad developeruntil satisfactory staining was observed. Staining was stopped with 5%acetic acid.

Non-carbohydrate aromatic polymer component:

DEAE Purification

11.7 mg of dialyzed Galahad ammonium sulfate precipitate were loadedonto a 2.5 cm wide by 1 cm high DEAE column equilibrated in pH 6.8 20 mMTris. Sample was loaded in 1.0 ml of equilibration buffer then washedwith 3.0 ml of same. The column was then eluted with 5 ml 20 mM Tris 1MNaCl producing a colorless eluate. Finally, color was eluted from thecolumn with 10 ml 25% NaOH. Eluate was dialyzed against RODI water andlyophilized to give a pink powder, SS120808. Note that some pink colorwas retained on column.

Butanol HCl Iron Digestion

Samples were treated according to the method of Porter and Chan(Phytochemistry (1986) v25(1) p223). Briefly, 3 mg of Galahad Ammoniumsulfate precipitate or green tea extract (positive control) weresuspended in butanol concentrated HCl 19:1 with 33 ul of 2% ferricammonium sulfate then incubated at 100 celsius for 40 minutes. Thisreaction is judged to be positive if an increase in absorbance at 650 nmis observed.

Extraction of Low Molecular Weight Proanthocyanidins

Low molecular weight anthocyanidins were extracted using theprodelphinidin gallate extraction procedure of Nishioka et al. (Nishiokaet al. Chem. Pharm Bulletin, v31(11) p3906-3914 1983) from 200 ul ofGalahad 08:016 in parallel with 2 g of Da Li Shu green tea using 80%acetone which was then dried to a minimal volume and filtered. Noprecipitate was noted upon addition of acetone to Galahad. Samples werethen extracted 3 times with 1 volume ethyl acetate. 0.2 mg of materialwas obtained from Galahad and approximately 200 mg from the green tea.

TLC was performed on Whatman HPTLC plates using eitherchloroform:methanol 4:1 or benzene:acetone:acetic acid 4:1:1 asdeveloping solvents. Plates contained a UV fluorescent dye and wereexamined for the presence of UV absorbing compounds under UVillumination then stained with Ceric ammonium nitrate ammoniummolybdate.

Isolated phloroglucinolysis products were analyzed by spotting onto amatrix of 2,5 dihydroxybenzoic acid. MS analysis of the products wasperformed on an Applied biosystems Voyager MALDI run in the positive ionmode.

UV/Visible Spectroscopy

Samples were suspended in 0.5M HCl in Methanol and spectra were recordedon a Beckman DU600 UV visible spectrophotometer. Periodate oxidation wasaccomplished by suspending sample in approximately 40 ul HIO₄ thendiluting solution into methanolic HCl for spectrophotometry.

CHN Analysis

Carbon Hydrogen Nitrogen Analysis was performed by the Chemical AnalysisLaboratory of the UGA.

Protein and Composition Analysis of Galahad Ammonium Sulfate Precipitate

Ammonium Sulfate Precipitation

The contents of 1 vial of Galahad were precipitated with 5 volumes ofsaturated ammonium sulfate. Sample was incubated on ice. Pellet washarvested by centrifugation.

Protein Assay

Samples were analyzed for protein using the Bio Rad Microplate proteinassay. Briefly, 10 ul of sample or BSA standard were incubated with 200ul Bio Rad reagent (diluted 1:4 with water) for 5 minutes at roomtemperature. Absorbance at 650 nm was monitored using a MolecularDevices plate reader.

Heptafluorobutyryl (HFB) Composition

Glycosyl composition analysis was performed by combined gaschromatography/mass spectrometry (GC/MS) of the HFB derivatives of themonosaccharide methyl glycosides produced from the sample by acidicmethanolysis.

Methyl glycosides were first prepared from dry sample provided by theclient by methanolysis in 1M HCl, in methanol at 80° C. (18-22 hours),followed by acetylation with heptafluorobutyric acid anhydride. GC/MSanalysis of the HFB methyl glycosides was performed on an HP 5890 GC,using a CPSil-5 Low Bleed capillary column (30m×0.25 mm ID).

TLC

The initial solvent used for TLC of intact Galahad Red was the strongsolvent used for LC—methanol:water:acetic acid 48:1:1. This was used todevelop silica gel Kieselgel 60 F254 plates which were examined forvisible TLC bands, illuminated with UV light to find bands absorbing inthe UV spectrum then stained with ceric ammonium nitrate ammoniummolybdate to locate other, non UV absorbing materials.

Ultrafiltration

To see whether Galahad Red is approximately the same size as thecompound characterized in the laser light scattering experiments, weultrafiltered 50 ul aliquots of Galahad lot 08:016 through filters of 10kD and 100 kD MWCO. Filtrates were inspected visually for color.

Phloroglucinolysis

Two different methods of phloroglucinolysis were used. For breakdown ofthe components of a standard compound, Carlo Rossi Burgundy, we usedincubation in 1M HCl in methanol at 80° C. for 2 hours in the presenceof 50 mg phloroglucinol. All hydrolyses of Galahad Red were made using50 mg/ml phloroglucinol in 1M HCl in methanol 80° C. with overnightincubation. Reactions were analyzed by TLC on silica gel TLC platesusing chloroform methanol 9:1 and in some cases chloroform methanol0.25% KCl 5:4:1 as a developing solvent. Visualization was accomplishedeither by the use of a UV lamp and by staining with ceric ammoniumnitrate/ammonium molybdate in ethanol sulfuric acid.

MALDI-Ms for Determination of Molecular Weight

Isolated phloroglucinolysis products were analyzed by spotting onto amatrix of 2,5 dihydroxybenzoic acid. MS analysis of the products wasperformed on an Applied biosystems Voyager MALDI run in the positive ionmode.

Purification of Galahad Red on Butyl Sepharose

200 ul of Galahad lot 08:016 were loaded on a 08×8 cm column of butylsepharose equilibrated in water. Column was then eluted with 5 ml ofwater giving some colored compounds but leaving most color at the top ofthe column. Column was then eluted with methanol, isopropanol,hexane:isopropanol 1:1, 1M methanolic HCl, n butanol, Isopropanol with1% TEA and 1% HOAc, pyridine, and phenol: 0.5M methanolic HCl 1:1. Onlythe phenol methanolic HCL eluted any significant color and a substantialamount of material was left on the column.

Amino Acid Composition

Amino acid composition analysis was performed by combined gaschromatography/mass spectrometry (GC/MS) of the heptafluorobutyrate(HFB) derivatives of the amino acid isoamyl esters produced from thesample by acidic transesterification as per the method of Pons et al.(Pons et al. Biochemistry 2003, v42 p 8342-8353).

Briefly, samples were first hydrolyzed in 6M HCl overnight at 110° C.lnmol of norleucine internal standard was then added and samples weredried under a gentle stream of nitrogen. Samples were then resuspendedin 0.5M methanolic HCl and incubated at 80° C. overnight.Transesterification was then performed in 1.5M isoamyl HCl at 100° C.overnight followed by evaporation under nitrogen and acylation withheptafluorobutyric acid anhydride (50 ul in 200 ul acetonitrile). GC/MSanalysis of the amino acid derivatives was performed on an HP 5890 GCinterfaced to a 5970 MSD, using a CP Sil5 capillary column (30m×0.25 mmID) and a temperature gradient of 90 to 260° C. at 5° C./min.

Biological Activity of Separated Material

CPE Assay

Human foreskin fibroblasts (HFF) cells were plated into six well platesand incubated at 37° C. with 5% CO₂ and 90% humidity. Two days later,drug was serially diluted 1:5 in MEM with 2% FBS using sixconcentrations of drug. The virus to be used was diluted in MEMcontaining 10% FBS to a desired concentration which gave 20-30 plaquesper well. The media was then aspirated from the wells and 0.2 ml ofvirus was added to each well in triplicate with 0.2 ml of media beingadded to control wells. The plates were then incubated for one hour withshaking every fifteen minutes and drug was added to appropriate wells.After an incubation period of 3 days, the cells were stained with 0.1%crystal violet in 20% methanol, washed with PBS, and the plaques countedusing a stereomicroscope. Plaque number in drug treated and untreatedwells, were used to calculate EC₅₀ values.

Direct Inactivation

Two days prior to use, HFF cells were seeded in six well plates andincubated at 37° C. On the day of the assay the virus to be used wasdiluted in MEM with 10% FBS to a desired concentration which would yield40-60 plaques. The drug was at an initial concentration of 100%solution. This was serially diluted 1:5 to give six concentrations in0.4 ml of media. Then 0.4 ml of virus was added to each concentration ofdrug to yield the final concentrations ranging from 50% to 0.016%, and0.4 ml of MEM with 10% FBS was added to the virus control and cellcontrol tubes. The mixtures were incubated in a 37° C. water bath for 1hour. The mixtures were then placed on ice. The media was aspirated fromthe wells of confluent HFF cells and 0.2 ml of the mixture was added tothe appropriate wells in duplicate. The plates were then incubated for 1hour at 37° C. and shaken every 15 minutes. After the incubation, 2.0 mlof MEM with 2% FBS and pooled human sera was added to each well. Theplates were then incubated for 3 days, after which the cells werestained with 1 ml per well of 0.1% crystal violet/20% methanol the wellswashed with PBS, and the plaques counted using a stereomicroscope. TheEC₅₀ values were calculated using a computer program.

Neutral Red Uptake Assay

Compound concentration that reduced the uptake of the neutral red vitaldye by 50% (CC₅₀) was used as a measure of toxicity. Briefly, 2.5×10⁴cells were seeded into each well of 96 well tissue culture platescontaining growth media and incubated for 24 h at 37° C. in a CO₂incubator. Media containing compound dilutions were then added to theplates, which were incubated for an additional 7 days. Cell monolayerswere stained with a 0.01% solution of neutral red in PBS and incubatedfor 1 hour. Cells were washed and dye internalized by the cells wassolubilized in 100 μl of a 50% ethanol solution supplemented with 1%glacial acetic acid and the optical density was determined at 540 nm.EC₅₀ values were calculated by standard methods (as per Kern, E. R., N.L. Kushner, C. B. Hartline, S. L. Williams-Aziz, E. A. Harden, S. Zhou,J. Zemlicka, and M. N. Prichard. 2005. In vitro activity and mechanismof action of methylenecyclopropane analogs of nucleosides againstherpesvirus replication. Antimicrob Agents Chemother 49:1039-45).

Test for Tannin, Purification and Semipreparative Phloroglucinolysis onGalahad Red

Tannin Test

500 ul of sample or Carlo Rossi Burgundy (positive control) were addedto 1 ml of 1 mg/ml BSA suspended in either 50 mM tricine pH 8.0 or pH5.0 ammonium formate and observed for precipitation. As a control,buffer without BSA was added.

Purification

20 ml of Galahad TK020209 were precipitated with 17 ml saturatedammonium sulfate. The deep red precipitate from centrifugation (15minutes 3000 rpm) was then suspended in 100 mM TFA pH 1.3 andrecentrifuged. Pellet was then dissolved in 40 ml water+1 mlconcentrated ammonia and recentrifuged. Little material was pelleted atthis stage and the supernatant was lyophilized to get 740 mg Galahad RedSS030909A.

Semipreparative Phloroglucinolysis

55 mg of Galahad Red SS030909a were suspended in 3 ml 50 mg/mlphloroglucinol 1M methanolic HCl and incubated overnight at 80° C.Reaction was then partially dried, diluted with 10volumes of water andloaded onto a 20 ml C18 sep pak (Waters). Column was then eluted with 30ml acetonitrile in 4 fractions, 10 ml 15% acetonitrile, then 20 ml 100%acetonitrile in 3 fractions. Significant color was noted in the first 2acetonitrile fractions. Column was then eluted with 8 ml benzeneisopropanol, 10 ml of same and 10 ml of same with 1% TFA. A brightyellow color was observed in benzene isopropanol wash 2 and a red colorin benzene isopropanol TFA wash.

Material eluting in acetontrile showed a series of bands by TLC inbenzene acetone acetic acid 5:4:1 (ceric ammonium nitrate stain). Halfof the eluate was purified by preparative TLC on a 20×20 cm AnaltechSilica gel H TLC plate. 7 different bands of material were scraped fromthe plate and eluted in methanol. These are numbered from highest Rf tolowest with 7 being material retained at the origin.

Mass Spectrometry MALDI-MS Both analyzed on an Applied Biosystems 4700MALDI TOF MS run in both the positive and negative ion modes using DHBas a matrix. Samples were spotted from methanol.

Phloroglucinolysis

Two different methods of phloroglucinolysis were used. For breakdown ofthe components of a standard compound, Carlo Rossi Burgundy, we usedincubation in 1M HCl in methanol at 80° C. for 2 hours in the presenceof 50 mg phloroglucinol. All hydrolyses of Galahad Red were made using50 mg/ml phloroglucinol in 1M HCl in methanol 80° C. with overnightincubation. Reactions were analyzed by TLC on silica gel TLC platesusing chloroform methanol 9:1 and in some cases chloroform methanol0.25% KCl 5:4:1 as a developing solvent. Visualization was accomplishedeither by the use of a UV lamp and by staining with ceric ammoniumnitrate/ammonium molybdate in ethanol sulfuric acid.

Isolation of Phloroglucinolysis Reaction Products

Reaction mixes were dry loaded onto columns of Iatrobeads for flashchromatography. Initial solvent was chloroform, followed by chloroformmethanol 9:1 then 4:1.

NMR

Proton NMR was acquired using a Varian Inova 600 MHz instrument at 25°C. using a 3-mm cryogenic probe. Spectra were recorded in CDCl₃ or CD₃ODas solvent. Chemical shifts were referenced to the respective residualsolvent signal (CDCl₃: 7.26 ppm, 77.16 ppm; CD₃OD: 3.31 ppm, 49.00 ppm)

Solid Phase Extraction (SPE) with Sep Pak C18

Galahad (070609) was lyophilized. Solid material was dissolved in 10 mMammonium acetate and loaded on Sep-Pak C18 catridge, followed by elutionwith 10 mM ammonium acetate, water and acetontrile as illustrated inScheme 1. The hydrophilic and hydrophobic fractions collected werelyophilized and analyzed by ¹H NMR.

Fractional Precipitation with Methanol and Iso-Propanol

Galahad (070609) was lyophilized. Solid material was dissolved in 10 mMammonium acetate and precipitated in 90% methanol. The methanol solublepart was evaporated, re-dissolved in 10 mM ammonium acetate andprecipitated in 80% iso-propanol as shown in Scheme 2. All fractionswere analyzed by GC/MS after TMS derivatization or by ¹H NMR.

NMR Spectroscopy of Monosaccharide Methyl Glycosides

Samples were dissolved in 0.2 mL D₂O or methanol-d4. 1D proton NMRspectra were acquired on a Varian Inova-600 MHz spectrometer at 298 K(25° C.) using standard Varian pulse sequences. Proton chemical shiftswere measured relative to internal acetone (δ_(H)=2.225 ppm). The samplewas dissolved in DMSO and again permethylated by the method of Ciukanuand Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodiumhydroxide and methyl iodide in dry DMSO). The sample was subjected tothe NaOH base for 10 minutes then methyl iodide was added and left for10 minutes. More methyl iodide was then added for 40 minutes. The basewas then added for 10 minutes and finally more methyl iodided was addedfor 40 minutes. This addition of more methyl iodide and NaOH base was toinsure complete methylation of the polymer. Following sample workup, thepermethylated material was hydrolyzed using 2 M trifluoroacetic acid (2h in sealed tube at 121° C.), reduced with NaBD₄, and acetylated usingacetic anhydride/trifluoroacetic acid. The resulting PMAAs were analyzedon a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selectivedetector, electron impact ionization mode); separation was performed ona 30 m Supelco 2330 bonded phase fused silica capillary column.

Results

Initial studies of Galahad focused on an arabinan which is present inthe sample. According to glycosyl composition analysis before and afterammonium sulfate precipitation the arabinan precipitates together withthe material producing the red color.

Glycosyl Compositon

TABLE D1 Glycosyl Composition Analysis of Galahad Sample Glycosylresidue Mass (μg) Mole %¹ Crude Rhamnose (Rha) 25.5  7.1 Galahad Fucose(Fuc) n.d. n.d. Arabinose(Ara) 141.0  42.9  Xylose (Xyl) 8.5 2.6Glucuronic Acid(GlcUA) 17.3  4.1 Galacturonic acid (GalUA) 103.1  24.3 Mannose (Man) 14.1  3.6 Galactose (Gal) 42.1  10.7  Glucose (Glc) 18.8 4.8 N Acetyl Galactosamine (GalNAc) n.d. n.d. N Acetyl Glucosamine(GlcNAc) n.d. n.d. Heptose(Hep) n.d. n.d. 3 Deoxy-2-manno-2 Octulsonicacid n.d. n.d. (KDO) Sum 370    100    Galahad Rhamnose (Rha) 0.9 2.108:016 Fucose (Fuc) n.d. n.d. A.S. Ppt Arabinose(Ara) 31.6  77.8  Xylose(Xyl) n.d. n.d. Glucuronic Acid(GlcUA) n.d. n.d. Galacturonic acid(GalUA) 4.7 8.9 Mannose (Man) 0.5 1.0 Galactose (Gal) 4.1 8.4 Glucose(Glc) 0.9 1.8 N Acetyl Galactosamine (GalNAc) n.d. n.d. N AcetylGlucosamine (GlcNAc) n.d. n.d. Heptose(Hep) n.d. n.d. 3 Deoxy-2-manno-2Octulsonic acid n.d. n.d. (KDO) Sum 43   100   

TABLE D2 Average of the Glycosyl composition data. Mol % of sugar in thecarbohydrate component: Sugar Low % High % Mean ± 3SD Arabinose(Ara)33.2 74.2 53.7 ± 20.5 Rhamnose (Rha) 0 9.3 4.2 ± 5.1 Xylose (Xyl) 0 4.41.7 ± 2.7 Glucuronic Acid(GlcUA) 0 7.3 1.7 ± 5.6 Galacturonic acid(GalUA) 3.4 35.8 19.6 ± 16.2 Mannose (Man) 0 6.0 2.9 ± 3.1 Galactose(Gal) 1.9 19.5 10.7 ± 8.8  Glucose (Glc) 0 12.5 5.4 ± 7.1

Glycosyl Linkage

TABLE D3 Glycosyl Linkage Analysis of Galahad (CCRC Code DK121806)Percentage Glycosyl Residue Present terminally linked arabinofuranosylresidue (t-Araf) 14.9% 2-linked arabinofuranosyl residue (2-Araf) 9.6%2-linked rhamnopyranosyl residue (2-Rhap) 0.3% 3-linked arabinofuranosylresidue (3-Araf) 3.2% terminally linked galactopyranosyl residue (t-Gal)2.1% 5-linked arabinofuranosyl residue (5-Araf) 15.8% 3-linkedglucopyranosyl residue (3-Glc)&2,4-linked 0.7% rhamnopyranosyl residue(2,4-Rhap) 2-linked glucopyranosyl residue (2-Glc) 1.2% 4-linkedmannopyranosyl residue (4-Man) 1.4% 3,5-linked arabinofuranosyl residue(3,5-Araf) 6.9% 2,5-linked arabinofuranosyl residue (2,5-Araf) 5.3%4-linked glucopyranosyl residue (4-Glc) 4.7% 2,3,5-linkedarabinofuranosyl residue (3,5-Araf)& 2,3,4- 27.0% linkedarabinopyranosyl residue (2,3,4-Arap) 4-linked galactouronic acid (4-galA) 1.5% 3,6-linked galactopyranosyl residue (3,6-Gal) 0.4%2,3,4,6-linked mannopyranosyl residue (2,3,4,6-Man) 0.7% 2,3,4,6-linkedgalactopyranosyl residue (2,3,4,6-Gal)&2,3,4- 2.1% linked galactouronicacid 2,3,4,6-linked glucopyranosyl residue (2,3,4,6-Glc) 2.2%

TABLES D4 Glycosyl Linkage Analysis of various lots in detail PercentageGlycosyl Residue Present terminally linked rhamnopyranosyl residue(t-Rha) 1.42 terminally linked arabinofuranosyl residue (t-Araf) 21.90terminally linked arabinopyranosyl residue (t-Ara) 0.24 terminallylinked xylopyranosyl residue (t-Xyl) 0.67 2 linked rhamnopyranosylresidue (2-Rha) 3.48 terminally linked manopyranosyl residue (t-Man)1.12 terminally linked glucopyranosyl residue (t-Glc) 4.35 3 linkedarabinofuranosyl residue (3-Araf) 5.25 terminally linkedgalactopyranosyl (t-Gal) 6.36 4 linked arabinopyranosyl residue or5-linked 14.71 arabinofuranosyl residue (4-Ara or 5-Araf) 4 linkedxylopyranosyl residue (4-Xyl) 0.71 2,4 linked rhamnopyranosyl residue(2,4-Rha) 2.81 2 linked manopyranosyl residue (2-Man) 4.40 2 linkedglucopyranosyl residue & 2-gulcuronic acid residue 0.18 (2-Glc & 2-GlcA)3 linked galactopyranosyl residue (3-Gal) 3.36 4 linked manopyranosylresidue (4-Man) 0.45 3,4 linked arabinopyranosyl residue or 3,5 linked6.62 arabinofuranosyl residue (3,4-Arap or 3,5-Araf) 4 linkedgalacturonic acid residue & 4 linked 4.41 galactopyranosyl residue(4-Gal A & 4-Gal) 2,4 linked arabinopyranosyl residue or 2,5 linked 1.07arabinofuranosyl residue (2,4-Arap or 2,5-Araf) 4-linked glucopyranosylresidue & 4-Glucuronic acid residue 4.73 (4-Glc & 4-GlcA) 2,3 linkedmanopyranosyl residue (2,3-Man) 1.62 6-linked galactopyranosyl residue(6-Gal) 1.17 2,3,4 linked arabinopyranosyl residue (2,3,4-Ara) 2.33 3,4linked glucopyranosyl residue (3,4-Glc) 0.47 2,6 linked manopyranosylresidue (2,6-Man) 0.69 4,6 linked glucopyranosyl residue (4,6-Glc) 0.244,6 linked galacturonic acid residue & 4,6 linked 0.11 galactopyranosylresidue (4,6-Gal A & 4,6-Gal) 3,6 linked galactopyranosyl residue(3,6-Gal) 5.16 TK070609 Terminally linked Rhamnopyranosyl residue(t-Rha) 1.7 Terminally linked Arabinofuranosyl residue (t-Araf) 11.6Terminally linked Fucopyranosyl residue (t-Fuc) 0.1 Terminally linkedArabinopyranosyl residue (t-Ara) 1.0 Terminally linked Xylopyranosylresidue (t-Xyl) 0.8 2 linked Rhamnopyranosyl residue (2-Rha) 3.5Terminally linked Manopyranosyl residue & 4 linked 2.6 Rhamnopyranosylresidue (t-Man + 4-Rha) Terminally linked Glucopyranosyl residue &Terminally 3.3 linked Glucuronic Acid residue (t-Glc + t-GlcA) 3 linkedArabinofuranosyl residue (3-Araf) 3.9 Terminally linked Galactopyranosylresidue & Terminally 8.7 linked Galacturonic Acid residue (t-Gal +t-GalA) 4 linked Arabinopyranosyl residue or 5 linked 14.6Arabinofuranosyl residue (4-Arap or 5-Araf) 4 linked Xylopyranosylresidue (4-Xyl) 1.1 2,3-Rhamnopyranosyl residue (2,3-Rha) 0.12,4-Rhamnopyranosyl residue (2,4-Rha) 2.4 2 linked Manopyranosyl residue(2-Man) 3.0 2 linked Glucopyranosyl residue & 2 linked Glucuronic Acid0.4 residue (2-Glc + 2-GlcA) 3 linked Galactopyranosyl residue (3-Gal)2.9 4 linked Manopyranosyl residue (4-Man) 1.1 3,4 linkedArabinopyranosyl or 3,5 linked Arabinofuranosyl 6.4 (3,4-Arap or3,5-Araf) 6 linked Glucuronic Acid residue & 6 linked Glucopyranosyl 0.5residue (6-GlcA + 6-Glc) 4 linked Galacturonic Acid residue & 4 linked7.5 Galactopyranosyl residue (4-GalA + 4-Gal) 2,4 linkedArabinopyranosyl or 2,5 linked Arabinofuranosyl 1.4 (2,4-Arap or2,5-Araf) 4 linked Glucuronic Acid residue & 4 linked Glucopyranosyl 7.3residue (4-GlcA + 4-Glc) 2,3 linked Manopyranosyl residue (2,3-Man) 0.96 linked Galactopyranosyl residue (6-Gal) 1.1 2,3,4 linkedArabinopyranosyl residue (2,3,4-Ara) 4.0 3,4 linked Galacturonic Acidresidue & 3,4 linked 1.2 Galactopyranosyl residue (3,4-Gal + 3,4-GalA)3,4 linked Glucuronic Acid residue & 3,4 linked 0.4 Glucopyranosylresidue (3,4-Glc + 3,4-GlcA) 2,4 linked Manopyranosyl residue (2,4-Man)0.1 2,4 linked Galacturonic Acid residue & 2,4 linked 0.9Galactopyranosyl residue (2,4-GalA + 2,4-Gal) 4,6 linked Manopyranosylresidue (4,6-Man) 0.1 4,6 linked Glucopyranosyl residue & 4,6 linkedGlucuronic 0.5 Acid residue (4,6-Glc + 4,6-GlcA) 4,6 Galacturonic acidresidue & 4,6-Galactopyranosyl residue 0.5 (4,6-GalA + 4,6-Gal) 3,6linked Galactopyranosyl residue (3,6-Gal) 4.1 TK081109 Terminally linkedRhamnopyranosyl residue (t-Rha) 1.8 Terminally linked Arabinofuranosylresidue (t-Araf) 16.6 Terminally linked Fucopyranosyl residue (t-Fuc)0.1 Terminally linked Arabinopyranosyl residue (t-Ara) 0.8 Terminallylinked Xylopyranosyl residue (t-Xyl) 0.6 2 linked Rhamnopyranosylresidue (2-Rha) 3.5 Terminally linked Manopyranosyl residue & 4 linked2.5 Rhamnopyranosyl residue (t-Man + 4-Rha) Terminally linkedGlucopyranosyl residue & Terminally 3.3 linked Glucuronic Acid residue(t-Glc + t-GlcA) 3 linked Arabinofuranosyl residue (3-Araf) 4.1Terminally linked Galactopyranosyl residue & Terminally 10.0 linkedGalacturonic Acid residue (t-Gal + t-GalA) 4 linked Arabinopyranosylresidue or 5 linked 15.1 Arabinofuranosyl residue (4-Arap or 5-Araf) 4linked Xylopyranosyl residue (4-Xyl) 0.7 2,3-Rhamnopyranosyl residue(2,3-Rha) 0.0 2,4-Rhamnopyranosyl residue (2,4-Rha) 2.4 2 linkedManopyranosyl residue (2-Man) 4.6 2 linked Glucopyranosyl residue & 2linked Glucuronic Acid 0.4 residue (2-Glc + 2-GlcA) 3 linkedGalactopyranosyl residue (3-Gal) 2.2 4 linked Manopyranosyl residue(4-Man) 0.5 3,4 linked Arabinopyranosyl or 3,5 linked Arabinofuranosyl5.4 (3,4-Arap or 3,5-Araf) 6 linked Glucuronic Acid residue & 6 linkedGlucopyranosyl 0.2 residue (6-GlcA + 6-Glc) 4 linked Galacturonic Acidresidue & 4 linked 7.5 Galactopyranosyl residue (4-GalA + 4-Gal) 2,4linked Arabinopyranosyl or 2,5 linked Arabinofuranosyl 0.7 (2,4-Arap or2,5-Araf) 4 linked Glucuronic Acid residue & 4 linked Glucopyranosyl 9.5residue (4-GlcA + 4-Glc) 2,3 linked Manopyranosyl residue (2,3-Man) 1.06 linked Galactopyranosyl residue (6-Gal) 0.7 2,3,4 linkedArabinopyranosyl residue (2,3,4-Ara) 1.8 3,4 linked Galacturonic Acidresidue & 3,4 linked 0.8 Galactopyranosyl residue (3,4-Gal + 3,4-GalA)3,4 linked Glucuronic Acid residue & 3,4 linked 0.2 Glucopyranosylresidue (3,4-Glc + 3,4-GlcA) 2,4 linked Manopyranosyl residue (2,4-Man)0.1 2,4 linked Galacturonic Acid residue & 2,4 linked 0.6Galactopyranosyl residue (2,4-GalA + 2,4-Gal) 4,6 linked Glucopyranosylresidue & 4,6 linked Glucuronic 0.3 Acid residue (4,6-Glc + 4,6-GlcA)4,6 Galacturonic acid residue & 4,6-Galactopyranosyl residue 0.4(4,6-GalA + 4,6-Gal) 3,6 linked Galactopyranosyl residue (3,6-Gal) 1.8TK020209 Terminally linked Rhamnopyranosyl residue (t-Rha) 2.2Terminally linked Arabinofuranosyl residue (t-Araf) 20.7 Terminallylinked Arabinopyranosyl residue (t-Ara) 1.1 Terminally linkedXylopyranosyl residue (t-Xyl) 1.3 2 linked Rhamnopyranosyl residue(2-Rha) 3.0 Terminally linked Manopyranosyl residue & 4 linked 2.5Rhamnopyranosyl residue (t-Man + 4-Rha) Terminally linked Glucopyranosylresidue & Terminally 6.0 linked Glucuronic Acid residue (t-Glc + t-GlcA)3 linked Arabinofuranosyl residue (3-Araf) 3.4 Terminally linkedGalactopyranosyl residue & Terminally 6.9 linked Galacturonic Acidresidue (t-Gal + t-GalA) 4 linked Arabinopyranosyl residue or 5 linked18.4 Arabinofuranosyl residue (4-Arap or 5-Araf) 4 linked Xylopyranosylresidue (4-Xyl) 1.0 2,4-Rhamnopyranosyl residue (2,4-Rha) 2.2 2 linkedManopyranosyl residue (2-Man) 4.6 2 linked Glucopyranosyl residue(2-Glc) 0.4 3 linked Galactopyranosyl residue (3-Gal) 2.9 4 linkedManopyranosyl residue (4-Man) 2.9 3,4 linked Arabinopyranosyl or 3,5linked Arabinofuranosyl 5.4 (3,4-Arap or 3,5-Araf) 2 linkedGalactopyranosyl residue & 6 linked Manopyranosyl 0.5 residue (2-Gal +6-Man) 4 linked Galacturonic Acid residue & 4 linked 2.4Galactopyranosyl residue (4-GalA + 4-Gal) 2,4 linked Arabinopyranosyl or2,5 linked Arabinofuranosyl 0.9 (2,4-Arap or 2,5-Araf) 4 linkedGlucuronic Acid residue & 4 linked Glucopyranosyl 3.5 residue (4-GlcA +4-Glc) 2,3 linked Manopyranosyl residue (2,3-Man) 1.1 6 linkedGalactopyranosyl residue (6-Gal) 1.1 2,3,4 linked Arabinopyranosylresidue (2,3,4-Ara) 1.9 3,4 linked Galacturonic Acid residue & 3,4linked 0.7 Galactopyranosyl residue (3,4-Gal + 3,4-alA) 4,6 linkedManopyranosyl residue (4,6-Man) 0.3 4,6 linked Glucopyranosyl residue &4,6 linked Glucuronic 0.3 Acid residue (4,6-Glc) 3,6 linkedGalactopyranosyl residue (3,6-Gal) 2.7

The glycosyl composition data shows that the Galahad carbohydrateportion is made of approximately 53% of arabinose, 19.6% of galacturonicacid, 10% of galactose, 5.4% of glucose, 4.2% of rhamnose, 1.7% ofxylose, 1.7% of glucuronic acid and 2.8% of mannose. The presence of thegalacturonic acid and glucuronic acid indicates that Galahad containsacidic residues as well as neutral monosaccharide residues.

The linkage analysis gives information on the type of glycosidiclinkages present in the polysaccharide. The glycosyl composition showedthat the carbohydrate portion of the sample is mainly arabinose with asmall amount of an unidentified residue and trace amounts of mannose,galactose, and rhamnose. These data suggest that the main carbohydratecomponent in the sample is an arabinan. The linkage data shows that thearabinan consist of a backbone of 5-Arabinose and 3,5-Arabinose withside chains of terminal arabinose and possibly 2-Arabinose backbone.

Size Exclusion Chromatography

The results of size exclusion chromatography are shown in FIG. 8.Approximate molecular weight of peaks was determined by comparison ofretention time to that of known standards. Molecular weight of the 41minute peak cannot be determined as it is in the included volume of thecolumn.

TABLE D5 Size determination of Peaks Retention Time Log M.W. (min) M.W.(kDa) Dextran Stds. 35.19 4.82 67 33.92 5.22 167 31.83 5.71 511 27.916.15 1400 Galahad 08:016 35.37 4.92 84

The red material within the sample was precipitated at the acidic pHused in the SEC. Thus, the molecular weight obtained by SEC likelyrefers to the arabinan.

NMR Spectroscopy

The 1-D proton NMR spectrum of Galahad showed a typical polysaccharidepattern with anomeric (5.2-4.5 ppm) and ring protons (4.3-3.5 ppm).Analysis of a set of 2-D spectra revealed several α-arabinofuranose spinsystems. β-Galactopyranose is a minor components in the sample. Apartial assignment is given in Table D7.

TABLE D7 NMR Chemical shift assignments of the arabinan. Chemical Shift(ppm) Residue 1 2 3 4 5 5′ t-α-Araf ¹H 5.21 4.17 3.99 4.08 3.85 3.74 ¹³C108.0 82.0 77.5 84.8 61.8 t-α-Araf ¹H 5.18 4.17 3.98 n.d. n.d. n.d. ¹³C107.8 82.0 77.5 n.d. n.d. 5-α-Araf ¹H 5.07 4.24 3.92 4.06 3.97 3.79 ¹³C108.2 82.0 77.5 82.0 3,5-α-Araf ¹H 5.10 4.16 4.07 4.24 3.97 3.83 ¹³C108.2 82.0 82.7 82.9 67.2 2,5-α-Araf ¹H 5.27 4.24 4.06 n.d. 3.97 3.83¹³C 107.3 86.2 n.d. n.d. 67.2 2,3,5-α-Araf ¹H 5.14 4.32 n.d. n.d. 3.973.83 ¹³C 108.2 86.2 n.d. n.d. 67.2 β-Galp ¹H 4.52 3.39 3.56 3.69 3.76n.d.

The 1-D spectrum also showed a broad peak around 7 ppm, indicatingpresence of an aromatic polymer (such as e.g. tannin). We attempted toremove this polymer by passage through a C18 column. It appeared thatthe fraction eluted with 5% acetic acid still contained the samerelative amount of aromatic polymer, while the fraction eluted withwater contained significantly less. It is not clear if the aromaticpolymer is covalently attached to the polysaccharide or if the twocomponents coelute from the column. It turned out that only a smallportion of the sample was eluted from the column.

The NMR results confirmed the linkage analysis data. Terminalarabinofuranose is the main component in the spectra, indicating thatthe sample is a highly branched arabinan.

Dynamic Light Scattering

To get an estimation of the size of the red material, which constitutesthe bulk of the product was obtained through light scattering. The DLSsoftware gave accurate molecular dimensions for the standards that werein agreement with the literature(http://www.dextran.net/dextran-physical-properties.html). The radiusvalues of the standards were fitted to a power trendline in Excel (seeFIG. 12), giving an R2 value of 0.9797, which indicates a good data fit.Both the raw Galahad sample and the ammonium sulfate precipitate eachshowed a major and a minor component. The minor component made up lessthan 5% of the mass in both samples. The measured radii of the twocomponents in both samples, as well as their mass average molecularweights, polydispersity, and mass contribution are tabulated in Table 3.

TABLE D6 DLS results Sample r (nm) % polydispersity MW (kDa) % massGalahad 6.1 13.0 68 >95 25.6 14.0 1,600 <5 AS ppt 5.4 24.6 54 >95 28.521.6 2,200 <5

The fact that the red material could be precipitated with ammoniumsulfate led us to believe that the material might include a protein.Amino acid analysis, however, gave us only very low levels of the twoamino acids, alanine and serine. Given these low levels of amino acidobserved and the fact that most proteins are composed of more than 2amino acids, Galahad Red is most likely composed of something other thanprotein.

This gives us a mass of around 68,000 for the most abundant component.We noted that Galahad Red could be precipitated by addition of ammoniumsulfate and could be separated from the carbohydrate material.

Molecular Weight Determination Using SDOC PAGE of Galahad and DEAEPurification

We have performed an initial carbohydrate gel electrophoresis analysisto check for polysaccharides. This analysis can tell us whether thecarbohydrate we have isolated by DEAE has a molecular weight consistentwith what we observe with our SEC analysis. It also allows us to checkfor the presence of saccharides in other fractions. SDOC of Galahadfractions showed that Galahad is mainly made of high molecular weightpolysaccharide.

High molecular weight saccharide was present in all fractions elutedfrom a DEAE column. The saccharide staining observed was consistent withthe 84 kDa saccharide seen by SEC analysis.

We have concluded that the molecular weight determination by SEC,Dynamic Light Scattering and gel electrophoresis all agree and indicatea high molecular weight species about 80 kD.

Butanol HCl Iron Digestion

Color reaction in the presence of acidic iron solution is often used asa test for tannin. The Galahad Red ammonium sulfate precipitate andgreen tea both produced the dark red color which is taken as a positivefor tannin, suggesting that Galahad Red is a high molecular weighttannin. Later experimenst led us to conclude that although the materialmay be a proanthocyanidin, it is not a tannin.

Extraction of Low Molecular Weight Proanthocyanidins

The results of this study are shown in FIG. 13.

MALDI TOF Mass spectrometry was used to analyze both intact andextracted Galahad as well as dowex desalted butanolic HCL iron digest.

TABLE D7 MALDITOF results of intact and extracted Galahad as well asdowex desalted butanolic HCL iron digest Predicted Sample Ion m/zObserved Ions Catechin 291 291 Procyanidin B1 578, 601 601 (M + Na)Galahad Extract 291, 295, 301, 303, 313, 317, 339, 361, 381, 409, 413,537, 545, 676, 699 Green Tea Extract 528 Galahad Iron Digest 215, 231,245, 317, 361, 375

Extraction of Galahad with ethyl acetate did not give enough material toanalyze by TLC. Acidic iron digestion suggested the presence of tanninin the ammonium sulfate precipitate. TLC shows both monomeric anddimeric cyanidins in the Green Tea extract.

UV/Visible Spectrophotometry

Spectra of Galahad compositions were compared to data obtained for knownanthocyanidins (Freitas and Mateus Environ. Chem. Letters (2006)4:175-183).

TABLE D8 UV/Visible Spectrophotometry Abs Abs Max Compound MaxGlycosylated Delphinidin 546 541 Petunidin 543 540 Malvidin 542 538Cyanidin 535 530 Peonidin 532 528 Pelargonidin 520 516 Galahad A.S. ppt468 Galahad DEAE 419 (shoulder at 500) Galahad DEAE post IO₄ ⁻ 370 (nodecrease on low side)

CHN Analysis

CHN analysis shows a high level of carbon relative to hydrogen in thesample and little nitrogen. This is consistent with an anthocyanidin orproanthocyanidin but inconsistent with protein, which would be expectedto have a high level of nitrogen.

TABLE D9 CHN analysis of the DEAE purified Galahad sample Sample % C % H% N Galahad DEAE 23.281 3.929 0.101

Protein and Composition Analysis of Galahad Ammonium Sulfate Precipitate

The results of protein quantitation are shown in Table 10.

TABLE D10 Protein Quantitation Average Concentration Absorbance ugProtein (mg/ml) Std Dev. Galahad Start 0.721 1.839416058 18.03 0.800.707 1.711678832 0.723 1.857664234 A.S. Ppt. 0.903 3.989484753 3.780.30 (10 mg/ml) 0.85 3.432176656 0.896 3.915878023

Note that both the Starting material and Ammonium sulfate precipitate(A.S. Ppt.) were originally assayed in a separate assay. Absorbance ofstarting material was above the level of the standard curve (BSA) andhad to be diluted prior to reanalysis. 1 μl of a 10 mg/ml solution ofammonium sulfate precipitate was assayed and 10 μl of starting materialdiluted 1:100 was also assayed.

The results of glycosyl composition analysis are given in Table D11 andare explained below. Threitol was added to the sample beforederivatization as an internal standard (10 micrograms to each sample).The sums of the area percentages shown here represent a sum of thevalues truncated to the first decimal place. There is thus a smallrounding error associated with the sum of the percentages which is whythey may not sum to 100.0%. Values under 2 ug or over 200 ug are givenas approximations only due to the lack of assay linearity in theseregions.

TABLE D11 Glycosyl Composition Analysis of A.S. Ppt. Sample MoietyGalahad 08:016 A.S. Ppt Mass (μg) Mole %¹ Rhamnose (Rha) 0.9 2.1 Fucose(Fuc) n.d. n.d. Arabinose(Ara) 31.6  77.8  Glucuronic Acid(GlcUA) n.d.n.d. Galacturonic acid (GalUA) 4.7 8.9 Mannose (Man) 0.5 1.0 Galactose(Gal) 4.1 8.4 Glucose (Glc) 0.9 1.8 N Acetyl Galactosamine (GalNAc) n.d.n.d. N Acetyl Glucosamine (GlcNAc) n.d. n.d. Heptose(Hep) n.d. n.d. 3Deoxy-2-manno-2 Octulsonic acid (KDO) n.d. n.d. 3OH C16 FA n.d. n.d. C16FA n.d. n.d. C18 FA n.d. n.d. Sum 43   100    ¹Values are expressed asmole percent of total carbohydrate. The total % carbohydrate iscalculated to be 6%. n.d. = none detected

Starting material is shown by this assay to have a very highconcentration of protein. One caveat to this is that we are using a dyebinding assay to measure protein and hydrophobic compounds can interferewith the assay, giving falsely high protein readings. Red color wasnoted to precipitate with ammonium sulfate. The ammonium sulfateprecipitate appears to be approximately 40% protein and 6% carbohydrate.The sugars observed in the precipitate are largely the same as thestarting material with only xylose absent (xylose was present at about 2mol % in the starting material). SDS PAGE was attempted to identifyproteins in this sample but low quantities of the red materialinterfered with the silver stain which we used to visualize the gel.Oddly, most of the red material aggregated at the top of the gel. Duringmethanolysis (used for HFB composition), the red material became muchmore soluble and could be seen to migrate on a silica gel TLC plate.

TLC

The results of TLC analysis of intact Galahad and phloroglucinolproducts is shown in FIG. 14.

The results of ultrafiltration of A.S. Ppt. through 2 filters is shownin Table D12.

TABLE D12 Ultrafiltration of A.S. Ppt. through 2 filters. Filtrate Color100 kD Pink  10 kD Clear

Mild phloroglucinolysis was ineffective in rendering the molecule intoits component anthocyanidin monomers, which suggested that it felllargely within the class of “condensed tannins” which are largelyunanalyzed. TLC on intact Galahad Red shows substantial material at theorigin. This, combined with the fact that ultrafiltration confirms thematerial to have a molecular weight over 10 kD mean that LC on silica ofthe intact material may well prove impossible. Our efforts to purifymaterial on butyl sepharose have fared little better with even extremelyhydrophobic solvents failing to elute all colored material. Our effortsat breaking up the molecule are meeting with more success.Phloroglucinolysis of the DEAE purified material shows all major bandsto be present in red wine meaning that we have no unusual anthocyaninadducts in the reaction products. Phloroglucinolysis of the ammoniumsulfate precipitate shows considerably more complexity with a number ofpotential partial phloroglucinolysis adducts present. MALDI MS of Pool2from the second phloroglucinolysis shows an ion with m/z 342, consistentwith the molecular mass of the sodium adduct of either petunidin orpeonidin (FIG. 15). As this is not a phloroglucinol adduct, it may wellbe that this residue is at one end of the polymer.

Amino Acid Analysis

Amino acid analysis indicated low levels of alanine and serine. The lowlevels of amino acid observed and the fact that most proteins arecomposed of more than 2 amino acids indicated that Galahad Red iscomposed of something other than protein.

Biological Activity of Separated Material

The majority of the biological activity resided in the ammonium sulfateprecipitate; the purified carbohydrate had little activity by itself:The results suggest that the combination of carbohydrate portion and theGalahad Red material to be biologically active.

TABLE D13 Biological Activity of Separated Components Direct Neutral RedCPE, Assay EC₅₀ Inactivation Uptake Component HSV-1 HSV-2 EC₅₀ CC₅₀ 1 *12.9 * 6.1       13 ± 0.5 **>4 2 92 48.2   51.5 ± 17 >1003 >100  >100    >100 ± 0 >100 4 >100  >100    >100 ± 0 >100 ^(A)5   0.05  0.01 **0.2 >0.4 ACV   1.4 1.8 NA >100 IGg NA    0.3 ± 0.3 NAComponents: 1. Ammonium sulfate pellet (SS101008a) 2. Ammonium sulfatesupernatant (SS101008b) 3. DEAE Load (SS101008c) 4. DEAE Eluate(SS101008d) (resupply) 5. Parent Compound (Galahad) * Drug color at 100μg/ml in CPE **Drug color at 100 μg/ml and 20 μg/ml. All Values are inμg/ml except #5 is % solution that is approximately 10 mg/ml^(A)Toxicity in the direct inactivation assay for #5 CC₅₀ is 5.5%.Galahad is %/ml for direct inactivation results and has an error ofabout 0.3

Given the color of the material and the fact that it did not seem to becomposed of protein, nucleic acid, or lipid, and had only low levels ofcarbohydrate, we consider that the material might be a polymericproanthocyanidin or tannin. We calculate that a polymer of the size wefind in light scattering should have around 200 monomers.

A classic test for tannin, using protein precipitation of BSA as amarker, was performed. Though our Gallo Burgundy standard precipitatedthe protein nicely, Galahad demonstrated no such precipitation ability.This indicates that although the material may be a proanthocyanidin, itis not be a tannin.

TABLE D14 Results of Tannin Test Precipitation with Low pH Neutral pHSample Low pH Neutral pH with BSA with BSA Galahad + − + − Carlo RossiBurgundy − − + +

Phloroglucinolysis

The results of TLC of a phloroglucinolysis reaction mix and fractionsfrom a C18 Column are shown in FIG. 16.

The results of TLC of a silica gel purification of an acetonitrile washfrom a C18 column are shown in Figure. 17.

MALDI MS

FIGS. 17-24 are representative spectra for most of the different silicagel chromatographic pools isolated above. No acceptable spectra wereobtained for pool 1, while pool 5 was deemed too impure for testing.Negative ion MALDI has been performed on many of the pools but requirescalibration before analysis.

TABLE D15 Proton NMR. Sample Chemical Shifts Silica Pool 3 7.07, 6.365,5.716, 1.293 Benzene isopropanol 8.71, 6.65, 6.16, 6.08, 5.92, 5.87,3.66(d), Eluate 3.37, 3.24, 1.80(m), 1.637(t), 1.435(m), 0.83(m),0.54(m) Benzene Ispropanol 6.56, 6.42, 6.07, 3.81, 3.73, 3.59, 3.53, TFAEluate 2.27(t), 1.401, 1.14(m), 0.896(m)

The test for tannin explains many things. The fact that the materialprecipitates at low pH explains why we could not analyze it by SEC—itprecipitated in the pH 5 buffer which we use for these assays. This alsoexplains why ammonium sulfate precipitates the material despite the factthat it is not a protein—the pH of ammonium sulfate is mildly acidic.This test also raises some questions: since Galahad Red does notprecipitate with the addition of protein, it cannot be classed as atannin. We also know that it is not a protein, nucleic acid,carbohydrate, or lipid.

We can get a better handle on this question by the use of a more highlypure preparation of Galahad Red than our previous purifications gave us.Washing the sample in TFA and resuspending in dilute ammonia gives us apreparation which shows much greater color in methanolic HCl than do theother preparations. Upon phloroglucinolysis, this material gives anarray of compounds (we now have a total of 9 pools of material) which issimilar to that of burgundy. The fact that Galahad Red responds tophloroglucinolysis suggests that the material is a non-tanninpolyflavone.

The MALDI-MS spectra indicates a complex set of mixtures indicating thatthe break down products are extremely complex. Of interest, in thebenzene iso-propanol eluate MS, the ion at m/z 556 is separated from themajor ion at 268 by 288 mass units, the mass of catechin.

NMR of both the benzene isopropanol eluates shows several interestingpoints. Both have chemical shifts between 6.0 and 6.1 ppm, consistentwith phloroglucinol but also have shifts at around 3.6 ppm, as expectedfor carbon 3 in the flavone skeleton. The benzene isopropanol eluatealso has shifts at 5.92 and 5.87 ppm corresponding to the expectedshifts of carbons 6 and 8 in the A ring of the flavone skeleton. Botheluates have shifts around 6.5 ppm, as expected for the B ring protonsof a flavone. Where these spectra differ from a standard flavone isaround 1 ppm where they have several multiplets which look almost like afatty acid. Note that we do not believe a fatty acid could be acontaminant of the preparation as it should have eluted in theacetonitrile wash. Esterification to one of the hydroxyls on a flavonering is ruled out by the harsh phloroglucinolysis conditions which wouldbe expected to transesterify the fatty acid, causing it to elute in theacetonitrile wash. It is possible that a long chain aldehyde could addto the flavone ring through an electrophilic substitution mechanism,forming a flavone adduct which would be highly hydrophobic (explainingthe extreme conditions required for elution from C18) and notsusceptible to hydrolysis. This type of addition has been documented inwine (Frietas and Mateus Environ Chem Lett (2006) 4:175-183 andreferences therein).

Glycosyl Composition Analysis of Galahad Red

The monosaccharides are identified by their retention times incomparison to standards, and the carbohydrate character of these areauthenticated by their mass spectra. For interpreting the mass spectraldata, fragment ion 73 is the characteristic base fragment for all TMSmethyl glycosides, 204 and 217 are characteristic of neutral sugars, and173 is characteristic of amino sugars. Fragment 217 is alsocharacteristic of uronic acids, and fragment ion 298 is characteristicof the sialic acids.

TABLE D16 Monosaccharides in Galahad Red Sample Glycosyl residue GalahadDEAE NaOH Mass (μg) Mole %¹ Arabinose(Ara) trace trace Ribose(Rib) n.d.n.d. Rhamnose (Rha) trace trace Fucose (Fuc) n.d. n.d. Xylose (Xyl)trace trace Glucuronic Acid(GlcUA) n.d. n.d. Galacturonic acid (GalUA)trace trace Mannose (Man) trace trace Galactose (Gal) n.d. n.d. Glucose(Glc) trace trace N Acetyl Galactosamine (GalNAc) n.d. n.d. N AcetylGlucosamine (GlcNAc) n.d. n.d. Heptose(Hep) n.d. n.d. 3 Deoxy-2-manno-2Octulsonic acid (KDO) n.d. n.d. Sum trace 100

All sugars in this assay were detected at less than 1 μg. This isoutside the linear range for our assay so we can only label them as“trace”. As 300 μg of material was loaded, the percentage of totalcarbohydrate in this sample is calculated to be less than 1%.

As proanthocyanidins are frequently glycosylated, we have analyzed theGalahad Red sample for carbohydrate. Carbohydrate analysis shows tracelevels of most of the sugars originally found in the sample. The factthat a variety of sugars are found but only in minute levels isconsistent with a low level of the previously characterizedpolysaccharides remaining in the current preparation. If any of theseresidues were utilized in glycosylation of the red material, we wouldexpect them to be present at much higher levels.

Phloroglucinolysis

Since anything more than such rudimentary analysis of the intactmaterial was not possible, we turned our efforts to degradation studies.Initially, we tried 3 different approaches; thiolysis, degradation usingbutanol/iron/acid and phloroglucinolysis. Phloroglucinolysis proved tobe the most informative assay. Initial conditions of hydrolysis werecomparatively harsh as we wanted to effect complete degradation of thepolymer. The results of TLC of the phloroglucinolysis reaction mix andfractions from a C18 column are shown in FIG. 16. When we went to a moremild phloroglucinolysis in the presence of ascorbic acid, we were ableto analyze the resulting compounds by HPLC, as shown in FIGS. 26-33.

Interestingly, the main peak observed is a catechin phloroglucinoladduct which is consistent with a polymer consisting of catechinsubunits. A small amount of nonconjugated catechin, which comes from thetermini of catechin chain, was also observed. No significant levels ofcompounds found in green tea (which is known to contain the antiviralcompound prodelphinidin gallate) were observed. While we can clearly seea catechin polymer in Galahad Red, we cannot prove that the polymer isexclusively catechin.

NMR Spectroscopy of Monosaccharide Methyl Glycosides

1D proton NMR spectra for fractions of Galahad, including GH-C18-1,GH-C18-2, GH-C18-3, GH-alcohol-1-MeOH insoluble, GH-alcohol-2-IPAinsoluble and GH-alcohol-3-IPA soluble, are shown and explained in FIGS.34-36.

The aromatic compounds are very likely glycosylated, which is why thesearomatic components have relatively high solubility in water. Theglycans attached (as in GH-alcohol-2 and GH-alcohol-3 or GH-C18-3) arearabinan with different linkages based on the 1D proton NMR spectra.

It is well known that both polyphenols (or other compounds with similarstructures) and arabinans have a variety of biological activities. Sofar more than 200 polyphenols have been identified in red wine. This isprobably why we always see broad peaks for the aromatic proton signalsin NMR spectra of original Galahad and all the fractions.

CONCLUSION

Galahad is made of two main polymers, which, in certain embodiments, areboth needed for full biological activity. Through various analyticaltechniques listed above we have concluded that Galahad is made of apolysaccharide portion and a non-carbohydrate polymer. The glycosylcomposition, linkage, and NMR analysis indicates that the maincarbohydrate component in the sample is an arabinan. Thenon-carbohydrate portion was indicated to be a catechin polymer. Anexample of a procyadin polymer is shown in FIG. 40. While we can clearlysee a catechin polymer in Galahad Red, we cannot prove that the polymeris exclusively all catechin.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. An inactivated anti-viral vaccine, the vaccinecomprising: (a) a composition that binds to a virus and inhibits saidvirus, said composition comprising: an isolated polysaccharidecomprising an arabinofuranosyl residue, a galactopyranosyl residue, anda galactouronic acid; and a catechin polymer; wherein said compositionis soluble in water; and wherein said composition is obtainable by amethod comprising: preparing a substantially homogeneous aqueous mixtureor solution of plant material from one or more plants of the Vitaceaefamily; contacting said mixture or solution with an ion exchange resinand recovering the colored product; and further purifying the coloredproduct by removing components that can pass through a dialysis filterthat generally passes molecules having a molecular weight of a 5 ×10⁵Daltons or less to produce said composition; and (b) the virus, whereinsaid composition binds to the virus and inactivates the virus, andwherein the vaccine is formulated as a unit dosage formulation.
 2. Aninactivated anti-viral vaccine, the vaccine comprising: (a) acomposition that binds to a virus and inhibits said virus, saidcomposition comprising: an isolated polysaccharide comprising anarabinofuranosyl residue, a rhamnopyranosyl residue, a galactopyranosylresidue, a glucopyranosyl residue, a mannopyranosyl residue, and agalactouronic acid; and a non-carbohydrate aromatic polymer; whereinsaid composition comprises about 40 to about 44 percent oxygen, about 44to about 48 percent carbon, about 3 to about 6 percent hydrogen; andabout 0.1 to about 1 percent nitrogen; wherein said composition issoluble in water; and wherein said composition is obtainable by a methodcomprising: preparing a substantially homogeneous aqueous mixture orsolution of plant material from one or more plants of the Vitaceaefamily; contacting said mixture or solution with an ion exchange resinand recovering the colored product; and further purifying the coloredproduct by removing components that can pass through a dialysis filterthat generally passes molecules having a molecular weight of a 5 ×10⁵Daltons or less to produce said composition; and (b) the virus, whereinsaid composition binds to the virus and inactivates the virus, andwherein the vaccine is formulated as a unit dosage formulation.
 3. Thevaccine of claim 1, wherein the composition comprises about 10 to about30 weight percent polysaccharide and about 70 to about 90 weight percentcatechin polymer.
 4. The vaccine of claim 1, wherein the compositioncomprises a component having a molecular weight greater than about 1million Daltons.
 5. The vaccine of claim 1, wherein the compositioncomprises a component having a molecular weight in the range of about60,000 to about 100,000 Daltons.
 6. The vaccine of claim 4, wherein thecomposition comprises an additional component having a molecular weightin the range of about 60,000 to about 100,000 Daltons.
 7. The vaccine ofclaim 6, wherein the ratio of the amount of the component having amolecular weight greater than about 1 million Daltons to the amount ofthe component having a molecular weight in the range of about 60,000 toabout 100,000 Daltons is about 95:5.
 8. The vaccine of claim 1, whereinsaid composition does not comprise protein.
 9. The vaccine of claim 1,wherein said polysaccharide comprises: about 30 to about 75 mole percentarabinose; about 0 to about 10 mole percent rhamnose; about 0 to about 5mole percent xylose; about 0 to about 8 mole percent glucuronic acid;about 3 to about 36 mole percent galactouronic acid; about 0 to about 6mole percent mannose; about 1 to about 20 mole percent galactose; andabout 0 to about 13 mole percent glucose.
 10. The vaccine of claim 2,wherein said polysaccharide comprises: about 60 to about 66 mole percentarabinose; about 4.1 to about 4.5 mole percent rhamnose; about 2.2 toabout 2.4 mole percent xylose; about 8.7 to about 9.7 mole percentgalactouronic acid; about 2.3 to about 2.5 mole percent mannose; about13.7 to about 15.1 mole percent galactose; and about 4.2 to about 4.6mole percent glucose.
 11. The vaccine of claim 1, wherein saidpolysaccharide comprises: a terminally linked arabinofuranosyl residue(t-Araf); a 2-linked arabinofuranosyl residue (2-Araf); a 2-linkedrhamnopyranosyl residue (2-Rhap); a 3-linked arabinofuranosyl residue(3-Araf); a terminally linked galactopyranosyl residue (t-Gal); a5-linked arabinofuranosyl residue (5-Araf); a 3-linked glucopyranosylresidue (3-Glc) and/or a 2,4-linked rhamnopyranosyl residue (2,4-Rhap);a 2-linked glucopyranosyl residue (2-Glc); a 4-linked mannopyranosylresidue (4-Man); a 3,5-linked arabinofuranosyl residue (3,5-Araf); a2,5-linked arabinofuranosyl residue (2,5-Araf); a 4-linkedglucopyranosyl residue (4-Glc); a 2,3,5-linked arabinofuranosyl residue(3,5-Araf) and/or a 2,3,4-linked arabinopyranosyl residue (2,3,4-Arap);a 4-linked galactouronic acid (4-gal A); a 3,6-linked galactopyranosylresidue (3,6-Gal); a 2,3,4,6-linked mannopyranosyl residue(2,3,4,6-Man); a 2,3,4,6-linked galactopyranosyl residue(2,3,4,6-Gal)&2,3,4-linked galactouronic acid; and a 2,3,4,6-linkedglucopyranosyl residue (2,3,4,6-Glc).
 12. The vaccine of claim 11,wherein said polysaccharide comprises: said terminally linkedarabinofuranosyl residue (t-Araf) comprises about 14.2 to about 15.7 wtpercent of said polysaccharide; said 2-linked arabinofuranosyl residue(2-Araf) comprises about 9.1 to about 10.08 wt percent of saidpolysaccharide; said 2-linked rhamnopyranosyl residue (2-Rhap) comprisesabout 0.3 wt percent of said polysaccharide; said 3-linkedarabinofuranosyl residue (3-Araf) comprises about 3.0 to about 3.4 wtpercent of said polysaccharide; said terminally linked galactopyranosylresidue (t-Gal) comprises about 2.0 to about 2.2 wt percent of saidpolysaccharide; said 5-linked arabinofuranosyl residue (5-Araf)comprises about 15.0 to about 16.6 wt percent of said polysaccharide;said 3-linked glucopyranosyl residue (3-Glc) and/or 2,4-linkedrhamnopyranosyl residue (2,4-Rhap) comprises about 0.7 wt percent ofsaid polysaccharide; said 2-linked glucopyranosyl residue (2-Glc)comprises about 1.1 to about 1.3 wt percent of said polysaccharide; said4-linked mannopyranosyl residue (4-Man) comprises about 1.3 to about 1.5wt percent of said polysaccharide; said 3,5-linked arabinofuranosylresidue (3,5-Araf) comprises about 6.6 to about 7.3 wt percent of saidpolysaccharide; said 2,5-linked arabinofuranosyl residue (2,5-Araf)comprises about 5.0 to about 5.6 wt percent of said polysaccharide; said4-linked glucopyranosyl residue (4-Glc) comprises about 4.6 to about 5.0wt percent of said polysaccharide; said 2,3,5-linked arabinofuranosylresidue (3,5-Araf) and/or 2,3,4-linked arabinopyranosyl residue(2,3,4-Arap) comprises about 25.7 to about 28.4 wt percent of saidpolysaccharide; said 4-linked galactouronic acid (4-gal A) comprisesabout 1.4 to about 1.6 wt percent of said polysaccharide; said3,6-linked galactopyranosyl residue (3,6-Gal) comprises about 0.4 wtpercent of said polysaccharide; said 2,3,4,6-linked mannopyranosylresidue (2,3,4,6-Man) comprises about 0.7 wt percent of saidpolysaccharide; said 2,3,4,6-linked galactopyranosyl residue(2,3,4,6-Gal)&2,3,4-linked galactouronic acid comprises about 2.0 toabout 2.2 wt percent of said polysaccharide; and said 2,3,4,6-linkedglucopyranosyl residue (2,3,4,6-Glc) comprises about 2.1 to about 2.3 wtpercent of said polysaccharide.
 13. The vaccine of claim 1, wherein thevirus is from a family selected from the group consisting ofAdenoviridae, Picornaviridae, Reoviridae, Arenaviridae, Bunyaviridae,Coroanviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae,Poxviridae Rhabdoviridae, Flaviviridae, and Retroviridae.
 14. Thevaccine of claim 2, wherein said composition provides a NMR spectrum asshown in FIG. 3A.
 15. The vaccine of claim 1, wherein said compositionis formulated in a delivery form selected from the group consisting of anasal spray and an injectable.
 16. The vaccine of claim 1, wherein thecomposition is obtained by said method comprising: preparing asubstantially homogeneous aqueous mixture or solution of plant materialfrom one or more plants of the Vitaceae family; contacting said mixturewith an ion exchange resin and recovering the colored product; andfurther purifying the colored product by removing components that canpass through a dialysis filter that generally passes molecules having amolecular weight of a 5 ×10⁵ Daltons or less to produce a compositionthat binds a virus.
 17. A method of preparing an inactivated anti-viralvaccine, said method comprising: contacting a virus with a compositionthat binds to the virus and inhibits said virus, said compositioncomprising: an isolated polysaccharide comprising an arabinofuranosylresidue, a galactopyranosyl residue, and a galactouronic acid; and acatechin polymer; wherein said composition is soluble in water; andwherein said composition is obtainable by a method comprising: preparinga substantially homogeneous aqueous mixture or solution of plantmaterial from one or more plants of the Vitaceae family; contacting saidmixture or solution with an ion exchange resin and recovering thecolored product; wherein said contacting is carried out for a sufficienttime to inactivate the virus.
 18. A method of inducing an immuneresponse in a mammal, said method comprising administering to saidmammal a vaccine according to claim 1 in an amount sufficient to inducean immune response.
 19. The vaccine of claim 1, wherein the vaccineadditionally comprises an adjuvant.
 20. The vaccine of claim 1, whereinthe vaccine does not comprise any adjuvant.
 21. The method of claim 18,wherein the method comprises administering the vaccine intranasally. 22.The method of claim 18, wherein the method comprises administering thevaccine by injection.
 23. The method of claim 22, wherein the methodcomprises administering the vaccine by intradermal injection.
 24. Themethod of claim 18, wherein the method comprises administering thevaccine in at least two, separate doses.